bluuebunny/crossref_metadata_2025 huggingface dataset:
NAADP mediated calcium release from acidic stores plays a critical role in downstream physiological responses of naïve CD4 and CD8 T cells (IRM10P.757)
Authors: Ali Ramadan , Camick Christina , Wiles Katherine , Slama James , Giovannucci Daivd , Wall Katherine
Published: January 2023

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger discovered to date and has been implicated in Ca2+ signaling in human Jurkat lymphoma T cells via ryanodine receptor activation. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naïve T cells has not been investigated. In the current study, we demonstrated for the first time to our knowledge the importance of NAADP in the generation of Ca2+ signals in murine conventional naïve T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the impact of NAADP on the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological endpoints such as proliferation, cytokine production, and other responses to stimulation of murine naïve T cells. Moreover, we demonstrated that acidic stores in addition to the endoplasmic reticulum are the relevant Ca2+ stores that were sensitive to NAADP in naïve T cells.
Prevention of enteric disease by peritoneal cells from Bacillus subtilis exopolysaccharide (EPS)-treated mice (MUC4P.840)
Authors: Jones Sara , Knight Katherine
Published: January 2023

Beneficial microbes modulate host immune responses and prevent pathogen-induced inflammation; the molecular mechanisms involved in this process are only recently being elucidated. The enteric pathogen Citrobacter rodentium shares genetic and phenotypic similarities to the human pathogen enteropathogenic E. coli. We previously demonstrated that probiotic B. subtilis, but not an exopolysaccharide (EPS) null mutant, protected mice from C. rodentium-associated disease. We also showed that purified EPS were sufficient to prevent disease when administered i.p. 24 hr prior to pathogen infection. In addition, we found that B. subtilis and EPS failed to protect TLR2 KO and TLR4 KO mice, suggesting that EPS-mediated protection utilizes both of these pathways. Since EPS bind peritoneal macrophages and because EPS are administered i.p., we hypothesized that peritoneal cells may contribute to protection. To test this idea, we adoptively transferred peritoneal cells from EPS-treated mice to naïve mice and found that these cells could prevent disease. We also show that TLR4 and TLR2 are required by peritoneal cells and non-peritoneal cells, respectively. We hypothesize that B. subtilis EPS mediate their protective effects through TLR4-expressing macrophages. Adoptive transfer studies with sorted cells from wt and TLR4 KO mice will be performed to test this idea.
The commensal bacteria metabolites short chain fatty acids positively regulate epithelial innate immune responses in the gut (MUC4P.847)
Authors: Kim Myunghoo , Kang Seung Goo , Park Jeong H. , Yanagisawa Masashi , Kim Chang H.
Published: January 2023

Short chain acids (SCFAs) are highly produced by gut commensal bacteria and play important roles in regulating immune responses. SCFAs can regulate epithelial cells an dimmune cells, and some of the functions are mediated through GPR41 and GPR43. Epithelial cells highly express both GPR41 and GPR43, but the functions of these receptors in regulation of epithelial innate immunity remain unclear. To investigate this, we utilized mice deficient in expression of GPR41 or GPR43. When the gut barrier function was breached with 50% ethanol, GPR41-/- and GPR43-/- mice were defective in expression of inflammatory cytokines (IL-6, IL1b and TNFa) and chemokines (CXCL1 and CXCL2) in response to invading bacteria. When the mice were infected with C. rodentium, the KO mice were delayed in mounting immune responses and clearing the pathogen. On the other hand, wild type mice with increased levels of SCFAs were more efficient in expression of the inflammatory factors. The results of our bone marrow transfer and in vitro culture experiments indicate that epithelial GPR41 and GPR43 play critical roles in mediating the SCFA signal. SCFAs induced a series of signaling events from the surface receptors to ERKs and to ATF2 activation to express the inflammatory factors. These processes are required to recruit and activate immune cells to clear pathogenic bacteria. Our results revealed a novel positive role of short chain fatty acids and their receptors in regulating intestinal immunity.
Vitamin A deficient mice have a decreased ability to clear the enteric pathogen, Citrobacter rodentium and increased mortality during infection (MUC4P.851)
Authors: McDaniel Kaitlin , Restori Katherine , Kennett Mary , Ross A. , Cantorna Margherita
Published: January 2023

Vitamin A is essential for normal immune function and vitamin A supplementation has been shown to decrease childhood mortality from infectious respiratory and diarrheal diseases. In this study, we determined that vitamin A sufficient (A+) mice were able to clear a Citrobacter rodentium infection while vitamin A deficient (A-) mice shed over 10,000 C. rodentium/g feces up to 37 d following infection. A- mice also had increased crypt hyperplasia compared to A- mice indicating that C. rodentium is a chronic infection in A- mice. Supplementation of A- mice with retinoic acid (RA) at d 0 or 14 post-infection resulted in the clearance of C. rodentium. A- mice had decreased frequencies of colonic CD8αα+ Tcrβ+ T cells and an increased frequency of CD4+ Tcrβ+ T cells. A- mice had reduced survival rates (58%) following C. rodentium infection while 100% of A+ mice survived. RA supplementation beginning on the day of infection resulted in 100% survival of the A- mice. The increased lethality of the C. rodentium infection in A- mice corresponded with the systemic spread of C. rodentium. T cells are critical for the prevention of the systemic spread and increased lethality of C. rodentium infection since mice without T cells are unable to clear an infection. The data suggest that vitamin A regulation of mucosal and systemic T cells is critical for survival and clearance of a C. rodentium infection.
The microbiome regulates the homeostasis of host invariant natural killer T (iNKT) cells (MUC4P.830)
Authors: An Dingding , Oh Sungwhan , Olszak Torsten , Neves Joana , Avci Fikri , Erturk-Hasdemir Deniz , Zeissig Sebastian , Blumberg Richard , Kasper Dennis
Published: January 2023

Symbiotic bacteria intimately participate in host physiological processes and impact host health and disease, although details at the molecular level largely remain unknown. We discovered that symbiotic Bacteroides species produce sphingolipids to modify the host antigen environment of the invariant natural killer T (iNKT) cells, a key immune modulator recognizing CD1d-restricted lipids. Using gnotobiotic technology and analytical tools, we found that bacterial glycosphingolipids, specifically one molecular fraction called GSL-Bf717, block iNKT cell activation and inhibit cell proliferation in early life. As a result, the total colonic iNKT cell number is restricted in adulthood and the host is protected when challenged by iNKT cell-mediated oxazolone colitis. These results suggest that intestinal bacterial sphingolipids are critical for establishing colonic iNKT cell homeostasis and host tolerance to environmental challenges. In addition, we find that the regulation depends on exposure to these symbiotic molecules in a critical early-life window, which, if missed, has irreversible impacts on host. This study contributes to our understanding of the essential role microbes play in host health and disease with precious molecular details that are largely lacking. The unique inhibitive power of GSL-Bf717 can be harnessed to provide therapeutic interventions in human autoimmune and allergic disorders where iNKT cell activation is detrimental.
Cadherin-11 regulates pulmonary fibrosis in bleomycin-induced lung injury (MUC5P.865)
Authors: Pedroza Mesias , George Teresa , Agarwal Sandeep
Published: January 2023

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible pulmonary disorder with unknown effective treatment. IPF is characterized by excessive myofibroblast differentiation in the alveolar airways. Epithelial-to-mesenchymal transition (EMT) is the differentiation of fibroblast-like phenotype from epithelial cells resulting in myofibroblast accumulation. Cadherin-11 (Cad11) expression on hyperplastic alveolar epithelial cells is a novel finding in our lab. Hence, we hypothesized that Cad11 contributes to the development of pulmonary fibrosis through EMT. Our aims consist of determining: 1) The role of Cad-11 in EMT; and 2) The contribution of Cad11 deficiency and neutralization in the intraperitoneal (IP) bleomycin (BLM) model of pulmonary fibrosis. Preliminary findings show that deficiency and neutralization of Cad11 in this model is associated with attenuated pulmonary fibrosis and myofibroblast accumulation. In vitro studies demonstrated that Fc-Cad-11 was able to stimulate MLE-12 cell lines to produce Col1α expression while knockdown of Cad-11 via siRNA in these epithelial cell lines prevented the increase of fibrotic markers. These findings demonstrate that Cad-11 contributes to the development and progression of pulmonary fibrosis. The novelty of this project will be the identification of mechanisms by which Cad11 regulates EMT. This project will support additional translational studies to develop Cad11 as a therapeutic target for pulmonary fibrosis.
Direct isolation of untouched human neutrophils from whole blood in as little as 25 minutes (TECH2P.870)
Authors: Tabatabaei-Zavareh Nooshin , Le Fevre Tim , Kokaji Andy , McQueen Karina , Fairhurst Maureen , Thomas Terry , Eaves Allen
Published: January 2023

Neutrophils are the first line of defense against microbial infections. They sense pathogen-associated molecular patterns through a wide range of receptors including toll-like receptors. Once activated, neutrophils eliminate infection via the phagocytosis of pathogens, releasing proinflammatory cytokines and antimicrobial peptides and producing reactive oxygen species (ROS). The procedures to enrich neutrophils from human whole blood involve at least two steps to remove red blood cells (RBCs) and unwanted leukocytes. For example, neutrophils may be separated from other leukocytes using density gradient centrifugation. However, they are still mixed with RBCs which are then lysed. Alternatively, RBCs are first depleted using lysis or sedimentation followed by fluorescence-activated cell sorting or magnetic cell separation to remove unwanted leukocytes. Here we describe a one-step immunomagnetic, column-free cell separation method which does NOT require RBC lysis, sedimentation or density gradient centrifugation. Non-neutrophils and RBCs are labeled with antibodies and cross-linked to magnetic particles. The labeled cells are removed using an EasySepTM magnet. The complete procedure takes 25-35 minutes. The purity of isolated neutrophils is 96±3% (n=14). The isolated neutrophils are functional and produce ROS upon activation with PMA. This fast isolation method of untouched, quiescent neutrophils facilitates the studies of neutrophil function in inflammatory diseases.
CD25 blockade with daclizumab alters regulatory T cell dynamics in RRMS patients (THER3P.880)
Authors: Huss David , Mehta Devangi , Sharma Akanksha , You Xiaojun , Riester Katherine , Sheridan James , Amaravadi Lakshmi , Elkins Jacob , Fontenot Jason
Published: January 2023

Regulatory T cells (Treg) mediate immune tolerance and depend on IL-2 for homeostasis and function. The biology of IL-2 is pleiotropic and complex. As a growth factor for both regulatory and conventional T cells, IL-2 is able to both potentiate and limit adaptive immune responses. Adding to this complexity, IL-2 can signal through a high-affinity (IL-2Rαβγ) and intermediate-affinity (IL-2Rβγ) receptor. Thus, the response to IL-2 at the single cell level is dependent on both receptor expression and local IL-2 concentration. Treg dysfunction is implicated in the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). Daclizumab, a humanized monoclonal antibody that binds the IL-2 receptor alpha chain and prevents its association with IL-2, has demonstrated efficacy in patients with RRMS, despite causing a reduction in Tregs. These seemingly contradictory observations led us to investigate Treg dynamics in placebo- and daclizumab-treated RRMS patients in the SELECT clinical trial. We report that CD25 blockade reduces Treg numbers, yet Tregs that remain preserve IL-2 signaling capabilities through the intermediate affinity IL-2Rβγ and maintain epigenetic marks characteristic of a phenotypically stable and functional Treg population. Furthermore, Treg dynamics did not associate with treatment efficacy or development of cutaneous adverse events, indicating that the effect of daclizumab on Treg dynamics may be independent of the therapeutic mechanism of action.
Improvement in treatment of experimental colitis in mice by using recombinant Lactococcus lactis with surface-displayed affibody against TNFα (THER4P.889)
Authors: Strukelj Borut , Perse Martina , Ravnikar Matjaz , Lunder Mojca , Cerar Anton , Berlec Ales
Published: January 2023

TNFα is one of the most studied pro-inflammatory cytokines in the pathology of irritative bovel disease (IBD), including Crohn's disease and ulcerative colitis. The current therapy for the treatment of mid-to severe IBD is composed by corticosteroids and parenteral administration of monoclonal antibodies against TNFα with systemic side effects. In order to develop an oral administration of anti-TNFα agent, we have introduced an affibody against TNFα as a binding molecule. A recombinant Lactococcus lactis was developed by means of expression of anti-TNFα affibody as a fusion protein with three functional parts: signal Usp45 protein for the secretion to the microenvironment in gut, TNFα-binding affibody domain and peptidoglycan-bynding AcmA autolysin protein that enables attachment of peptidic construct to the surface of L. lactis. Such recombinant bacteria were characterized by the capability to bind Alexa Fluor 488-conjugated TNFα. By means of flow cytometry we clearly demonstrated a distinct shift in fluorescence between control cells and TNFα-binding cells. Furthermore, mice model with induced experimental colitis was used for animal studies. After oral administration of wild type L. lactis (control group) and recombinant anti-TNF α L. lactis, we observed significantly decrease in the concentration of TNFα in the stool as well as general mucosal improvement in group treated with recombinant anti-TNF lactic acid bacteria.
PIKfyve, a class III lipid kinase, is required for TLR-induced type I IFN production via modulation of ATF3 (THER4P.890)
Authors: Cai Xinming , Xu Yongyao , Kim You-Me , Loureiro Joseph , Huang Qian
Published: January 2023

Type I IFN plays a key role in anti-viral responses. It also has been shown that deregulation of Type I IFN expression following abnormal activation of TLRs contributes to pathogenesis of systemic lupus erythematosus. In this study we find PIKfyve, a class III lipid kinase, is required for endolysosomal TLR-induced expression of type I IFN in mouse and human cells. PIKfyve binds to phosphatidylinositol 3P and synthesizes phosphatidylinositol (3,5)P2, and plays a critical role in endolysosomal trafficking. However, PIKfyve modulates type I IFN production via mechanisms independent of receptor and ligand trafficking in endolysosomes. Instead, pharmacological or genetic inactivation of PIKfyve rapidly induces expression of transcription repressor ATF3, which is necessary and sufficient for suppression of type I IFN expression by binding to its promoter and blocking its transcription. Thus, we have uncovered a novel phosphoinositide-mediated regulatory mechanism that controls TLR-mediated induction of type I IFN, which may provide new therapeutic indication for PIKfyve inhibitor.
Histological characterization of tumor-associated macrophages in patients with different stages of hepatocellular carcinoma (HCC) (TUM4P.914)
Authors: Tsuchimoto Yusuke , Asai Akira , Yokohama Keisuke , Ohama Hideko , Suzushi Tetsuya , Fukunishi Shinya , Tsuda Yasuhiro , Higuchi Kazuhide , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Tumor-associated macrophages, major leukocytes detected in tumor tissues, have been characterized as M2Mφ. Recently, M2Mφ have been classified into different subsets; M2aMφ, M2bMφ and M2cMφ. Therefore, in this study, the subset of M2Mφ present in tumor tissues of HCC patients was determined. Tumor tissues were obtained by liver biopsy and liver resection from patients with early and advanced stages of HCC. Paraffin embedded tissue specimens were stained for PE-conjugated anti-CD68 Ab. Also, the same tissue specimens were stained for FITC-conjugated anti-IL-12 Ab, PE-conjugated anti-IL-10 Ab, FITC-conjugated anti-CCL1 Ab, and FITC-conjugated anti-CD206 Ab. Stained histologic specimens were examined microscopically. In the results, large numbers of CD68+cells were predominantly present in the edge of the tumors. Large numbers of Mφ were not isolated from the tumor nests. CD68+cells isolated from advanced stage HCC patients were determined as IL-10+CD206+ cells, while a few numbers of these cells were demonstrated in the tumor tissues of early stage HCC patients. IL-10+CCL1+ cells and IL-12+CD68+ cells were not isolated from both advanced stage and early stage HCC patients. These results indicate that a majority of M2Mφ present in advanced stage HCC patients are M2aMφ and/or M2cMφ.
Role and association of CD11b and Gr1+ cells in promotion of spontaneous lung cancer metastasis (TUM4P.920)
Authors: Sharma Rajesh , Chheda Zinal , Jala Venkatakrishna , Satpathy Shuchismita , Haribabu Bodduluri
Published: January 2023

Microenvironment plays an important role in tumor growth and metastasis. We established a unique metastasis model by in vivo passaging the Lewis lung carcinoma (3LL) cells with 7-11 days of culture in between passages. When injected subcutaneously the parental (3LL) cells have minimal metastasis whereas the in vivo passaged (p3LL) cells showed robust metastasis. The goal of this study is to identify the immune population that enhances the metastatic propensity of tumor cells. Purified p3LL (CD45-) cells also yielded similar metastasis excluding a direct role of residual immune cells in promotion of metastasis. The p3LL cells metastasized at similar frequency in WT and Rag2-/- mice indicating that adaptive immune cells are not required for metastasis. Analysis of tumors derived from 3LL vs p3LL revealed differential accumulation of CD11b+Gr1+ subpopulations. 3LL tumors contained higher CD11b+ Gr1high cells whereas p3LL tumors showed higher levels of CD11b+ Gr1low population. Interestingly, when CD11b+ Gr1low populations were isolated from spleen of p3LL tumor bearing mice and co-cultured with 3LL cells for 5 days, these cells acquired enhanced metastatic capacity as compared to control 3LL cells. These results indicate a distinct association and a critical role of CD11b+ Gr1low population in promotion of metastatic phenotype and suggest that abundance of CD11b+ Gr1low population may serve as a marker and a potential therapeutic target for inhibiting Lung cancer metastasis.
Activation of dendritic cells by soypeptide lunasin as a novel vaccine adjuvant (VAC6P.942)
Authors: Tung Chun-Yu , Flores Sarah , Han Ling , Yao Shuyu , Zhou Baohua , Sun Jie , Chang Hua-Chen
Published: January 2023

Adjuvants enhance the immunogenicity of vaccines and improve the immune responses. FDA approved aluminum is known to promote Th2 immune response, which is the most used adjuvant in human vaccines; however it is less efficient on eliciting cellular immune responses. Thus, any agent capable of modulating the immune responses is the key to develop efficacious vaccines. We have identified a new use of soypeptide lunasin as a novel immune modulator. The objective is to define the effectiveness of lunasin as an adjuvant on activating immune responses. Human peripheral blood mononuclear cells of normal donors were stimulated with various concentrations of lunasin followed by phenotypic analysis of DC subsets. Lunasin-treated conventional DCs (cDCs) not only expressed elevated co-stimulatory molecules (CD86) but up-regulated chemokines (CCL2, CCL3, CCL4) and cytokine (IL-1β). Lunasin-treated cDCs induced higher proliferation of allogeneic CD4+ T cells when comparing with medium control treatment in mixed lymphocyte reaction. In addition to cDCs, pDCs responded to lunasin stimulation as evidenced by IFNa production. Lunasin administration increased CD86 expression in cDCs and induced IFNa production by pDCs in mice. Moreover, immunization with OVA and lunasin in mice improved survival against OVA-expressing influenza virus. Our data suggest that lunasin may act as an adjuvant by targeting both cDCs and pDCs to enhance and modulate immune responses to vaccine antigens.
Parvovirus-mediated aplastic crisis and the virus-specific immune response in patients with sickle cell disease (VAC6P.948)
Authors: Penkert Rhiannon , Tang Li , Thomas Paul , Rosch Jason , Hurwitz Julia , Hankins Jane
Published: January 2023

Parvovirus infection, which is typically subclinical in healthy individuals, can have catastrophic effects in patients suffering from sickle cell disease (SCD). Infection disrupts erythropoiesis, often resulting in aplastic crisis and other life-threatening complications in patients with SCD. Attempts to develop a safe and effective parvovirus vaccine have yet to be successful and may be improved by a better understanding of natural immune responses to parvovirus infections. St. Jude Children's Research Hospital houses one of the largest centers for pediatric SCD in the United States and is therefore uniquely positioned to monitor immune functions in patients with SCD. Here, we describe a retrospective analysis of patient records, examining parvovirus-specific antibody results from a semi-quantitative, commercially-available ELISA as a function of time relative to aplastic crisis. Results showed: (i) frequencies of seropositive immune responses in patients with SCD of various age groups were comparable to those of healthy controls, (ii) parvovirus-specific immune responses were rapidly induced in patients following aplastic crisis, and (iii) responses were generally sustained for years after the aplastic crisis event. Ongoing studies include further dissection of adaptive and innate immune responses toward parvovirus infections in this patient population to help define correlates of protection against parvovirus disease.
Parasite-neutralizing and protective antibody response in macaques immunized with synthetic LbL microparticle vaccines containing the conserved T1BT* epitope of Plasmodium falciparum (VAC7P.976)
Authors: Powell Thomas , Tang Jie , Jacobs Andrea , DeRome Mary , Palath Naveen , Cardenas Edwin , Boyd James , Nardin Elizabeth , Kaba Stephen , Lanar David
Published: January 2023

We have previously shown that synthetic LbL-MP vaccines containing the conserved T1B repeat and T* epitopes from the P. falciparum circumsporozoite (PfCS) protein elicit a protective antibody response in mice. Immunogenicity and efficacy of these LbL-MP were tested in a non-human primate model, the rhesus macaque. LbL-MP were loaded with T1BT* peptide with or without covalently-linked Pam3Cys, a TLR2 ligand. Both constructs elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFNγ secretion with little or no IL-5 secretion. The Pam3Cys-modified construct was more potent, generating antibody responses that neutralized wild-type P. falciparum in an in vitro hepatocyte invasion assay. In addition, IgG was purified from the macaque immune sera and passively administered to naïve mice that were then challenged with 5,000 transgenic P. berghei sporozoites that expressed the full-length PfCS protein. IgG from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected mice from challenge, with 50-88% of mice parasite-free for ≥ 15 days. These results demonstrate that synthetic LbL-MP vaccines bearing the conserved T1BT* epitopes of PfCS can elicit protective antibody responses against P. falciparum in a non-human primate model.
Efficacy of a vaccine for Angiostrongylus costaricensis against rat lungworm disease caused by A. cantonensis in wild Hawaiian Rats (Rattus rattus) (VAC7P.978)
Authors: Jarvi Susan , Pitt Will , Osuna Antonio , Farias Margaret , Shiels Laura , Howe Kay , Jacquier Steven , Shiels Aaron , Amano Karis , Luiz Blaine , Maher Daisy , Allison Maureen , Holtquist Zachariah , Scheibelhut Neil , Severino Michael , Riddick LaTasha
Published: January 2023

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen which causes a global, emerging infectious disease known as rat lungworm disease (RLWD). RLWD has been reported in 30 countries, and Hawaii is the epicenter for RLWD in the United States. We conducted a study in wild Hawaiian rats (Rattus rattus) to determine the efficacy of a vaccine developed against a related species (A. costaricensis). Twenty-eight wild adult rats were captured from Waiakea Forest Reserve on the Island of Hawaii. Rats were mated and 41 F1 offspring were vaccinated with two doses of PP2A vaccine, a serine/threonine phosphatase 2A recombinant peptide (4 µg vaccine and 4 µg adjuvant/25 g body weight) at >3 mos. of age. Rats were gavaged with 50 L3 stage larvae at four weeks post-vaccination. Rats were euthanized and necropsies conducted at 51-53 days PI. Spleen weight, spleen length, and lung weight were recorded and number of worms in heart and lungs counted. Adult worms were found in all F1 rats. An average of 20.88 worms/rat reached adulthood with an infective dose of 50 L3 stage larvae. We found no significant differences between vaccinated rats (n=21) and unvaccinated rats (n=20) in spleen weight (p=0.963), spleen length (p=0.830), lung weight (p=0.830) or number of worms collected from the heart and lungs (p=0.882), suggesting the vaccine does not provide adequate protective immunity to guard against infection by A. cantonensis in wild Hawaiian rats.
Rectal immunization with an rVCG vaccine protects against genital Chlamydia challenge (VAC7P.962)
Authors: Pais Roshan , Jha Rajneesh , Omosun Yusuf , He Qing , Fujihashi Kohtaro , Black Carolyn , Igietseme Joseph , Eko Francis
Published: January 2023

Pelvic inflammatory diseases consequent to Chlamydia trachomatis infections is a major public health challenge with frequent asymptomatic infections precluding in early diagnosis and treatment making clinical presentation of sequelae often the first indication of infection. Development of a safe, efficacious and affordable vaccine holds promise for the global spread of Chlamydia genital infections. Previously we have shown that intramuscular(IM) vaccination of Vibrio cholerae ghosts (VCG) expressing Porin B and PmpD showed protection of mice against intravaginal infections via the development of a cellular ( Th1) as well as humoral ( IgG2a and IgA) immunity. The present study was undertaken to examine the potential of rectal immunization for the induction of genital tract immunity against heterologous Chlamydia infection in mice. The impact of immunization with the VCG vaccine in combination with Flt3 Ligand (FL) on induction of immunity was also investigated. The results demonstrate that both IM and IR rVCG immunizations elicited high levels of antigen-specific Th1 cell-mediated and humoral immune responses that were enhanced following co-immunization with FL. Also, rectally immunized mice were significantly protected against heterologous infection with serovar E Chlamydia 2 weeks post immunization. These results highlight the potential of rectal immunization for eliciting immunity in the female genital tract against Chlamydia and other sexually transmitted diseases.
Phenotypic and functional evaluation of T cell responses after intradermal immunization with LTR192G, an attenuated form of the enterotoxigenic E. coli (ETEC) labile toxin (LT), as an adjuvant for an ETEC vaccine (VAC8P.1004)
Authors: Laird Renee , Gutierrez Ramiro , Savarino Stephen , Riddle Mark , Maciel Milton
Published: January 2023

The skin is an appealing route for delivery of vaccines. It is easily accessible, is rich in antigen presenting cells (i.e. dendritic cells), and several reports have shown that intradermal (ID) vaccination can use less antigen (dose sparing). However, few new adjuvants have been developed and tested for skin immunization. Here, we report our findings on the evaluation of the T cell response after immunization of volunteers with two prototype vaccines against ETEC given ID with LTR192G. Volunteers were immunized on days 0, 21 and 42 with the tip adhesin of the class 5a CFA/I fimbriae, CfaE, or an adhesin-toxoid chimera protein, CfaE-sCTA2/LTB5, admixed with the adjuvant. We found marked activation of LTB- and CfaE-specific CD4+ T cells post-immunization as measured by CD69 upregulation. Within the LTB-specific CD4+ population, we found a greater frequency of skin-homing CCR4+ cells, which is consistent with the route of immunization and a strong response to LTR192G. In addition, there was a higher frequency of LTB-specific CD4+ T cells with an effector memory phenotype. The frequency of CfaE-specific IFNγ+ CD4+ T cells was significantly higher post-immunization and was validated by high levels of IFNγ in CfaE-stimulated supernatants. Cytokine analysis also revealed higher levels of CfaE-specific IL-6 secretion compared to LTB. Taken together, the data demonstrate a strong CD4+ T cell response was generated to both antigen and adjuvant after ID immunization.
Inclusion of flagellin increases antibody responses in nonhuman primate neonates following influenza vaccination (VAC8P.1002)
Authors: Kim Jongrok , Holbrook Beth , Hayward Sarah , Blevins Lance , Alexander-Miller Martha
Published: January 2023

Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response to vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for use in this vulnerable population. To address this issue we developed an African Green Monkey model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a TLR5 agonist, to serve as an effective adjuvant for vaccination of neonates. Animals were primed and boosted with inactivated PR8 influenza virus in the presence of either flagellin or an inactive form of the protein. Inclusion of flagellin significantly increased the level of influenza-specific serum IgG present following boosting as well as at early times after virus challenge. Animals receiving flagellin adjuvanted vaccination also exhibited higher levels of respiratory influenza-specific IgG at early times post challenge. Interestingly, there was no flagellin mediated increase in IgM. The presence of flagellin also resulted in an increase in neutralizing antibody. This study supports further exploration of flagellin as an effective adjuvant for inducing vaccine responses against influenza in neonates.
Sepsis increases susceptibility to chronic infection and exacerbates CD8 T cell exhaustion (VIR4P.1008)
Authors: Condotta Stephanie , Rai Deepa , Griffith Thomas , Badovinac Vladimir
Published: January 2023

Sepsis survivors often display impairment in innate and adaptive immune responses and become highly susceptible to ‘secondary’ infections with intracellular pathogens that are normally controlled by CD8 T cells. It is unknown if or when this observed immunoparalysis of CD8 T cell immunity recovers, and the long-term consequences of sepsis on the ability of naïve CD8 T cells to respond to subsequent infections are poorly understood. Previously, we demonstrated that sepsis has sustained detrimental effects on the capacity of CD8 T cells to respond to acute viral and bacterial infections. Here, using the cecal ligation and puncture (CLP) mouse model, we show that mice recovering from a septic event are more susceptible to chronic LCMV Clone-13 infection demonstrated by increased morbidity and mortality. LCMV-specific CD8 T cells from chronically infected CLP mice display an increase in inhibitory molecule expression (e.g., PD-1) and diminished Ag-driven cytokine production, suggesting that sepsis exacerbates CD8 T cell exhaustion following chronic viral infection. Furthermore, this exhausted phenotype of virus-specific CD8 T cells is observed earlier in LCMV Clone-13 infected septic mice compared to control mice. Together, these results illustrate that sepsis increases the vulnerability to chronic infections and can result in substantial and long-lasting changes in the CD8 T cell compartment affecting the ability of the host to respond to newly introduced antigens.
Systemic hematogenous maintenance of memory inflation by MCMV infection (VIR4P.1013)
Authors: Smith Corinne , Turula Holly , Snyder Christopher
Published: January 2023

Cytomegalovirus (CMV) is a β-herpesvirus that establishes a latent infection and supports the life-long maintenance of very large numbers of virus-specific effector T cells. These T cells remain functional and are found in many organs in the body. Current models propose that, to sustain these populations, reservoirs of viral antigen and/or latently infected cells in lymph nodes stimulate T cell proliferation and differentiation, followed by migration of progeny to non-lymphoid tissues. We tested this model using murine CMV (MCMV). While MCMV transcriptional activity during latency was below our limit of detection, proliferation of MCMV-specific effector T cells was antigen-dependent and was used as a read-out for antigen encounter. As shown previously, T cells within draining lymph nodes divided at a higher rate than cells elsewhere. However, pulse-chase experiments with BrdU failed to reveal migration of recently divided T cells from lymphoid to non-lymphoid tissues. In fact, inflationary cells observed in non-lymphoid organs were primarily perfusion-resistant cells residing in the vasculature. Strikingly, inhibition of T cell egress from lymph nodes did not eliminate T cell division in circulation, and did not prevent the maintenance of the circulating inflationary population during long-term treatment. Together these results suggest that memory inflation in response to MCMV is driven by viral antigen presented by cells that are accessible to the blood supply.
Mouse cytomegalovirus glycoprotein m153 binds a dendritic cell-surface ligand (VIR5P.1026)
Authors: Tilahun Mulualem , Revilleza Maria Jamela , Mans Janet , Natarajan Kannan , Margulies David
Published: January 2023

Mouse cytomegalovirus (MCMV) is a valuable model for studying virus-host interactions and mechanisms of viral immune evasion. MCMV encodes the m145 family of glycoproteins with predicted MHC-I structure. Among these, m153 is expressed on the surface of infected cells predominantly during the early phases of infection, independent of β2 microglobulin and peptide. We previously determined the X-ray structure of m153, establishing that it has an MHC-I-like fold and forms a stable non-covalent dimer. To identify immune cells that might express a ligand for m153, we constructed a reporter cell in which the extracellular domains of m153 were linked to human ζ chain driving green fluorescent protein upon activation. Using the reporter cell assay, we observed that CD11c+ splenic dendritic cells (DCs) and bone marrow DCs potently stimulate the m153 reporter cell. Further characterization of these cells by flow cytometry staining with an m153 tetramer and a battery of DC markers, as well as antibody blocking experiments, suggested several candidates for the m153 ligand. Transfectant cells and gene knockout animals are being studied to confirm the identity of these candidates. The identification of a host cell ligand for m153 should improve our understanding of mechanisms of host immune suppression during viral infection and will provide insight on the evolution of viral immune evasion.
In vivo silencing of microRNA-155 (miR-155) ameliorated pulmonary hemorrhage induced by pristane (BA12P.100) (BA12P.100)
Authors: Zhou Shiyu , Liang Dong , Huang Xinfang , Harley John , Shen Nan
Published: January 2023

Background: MiR-155 is a typical pleiotropic miRNA that participates in various aspects of immunity. In this study, we systemically examined the role of miR-155 in the development of pulmonary hemorrhage (PH) in Pristane-induced lupus. Methods: Mice received a single i.p injection of 0.5 ml Pristane. 2 weeks later, lung tissues were collected for pathologic assessment. Total lung RNAs were assayed by qPCR or subjected to gene profiling. Gene profiling data was analyzed by IPA software. Serum cytokines were messured by Bio-plex Assays. Results: We showed that miR-155 was elevated in lung tissues after Pristane treatment. MiR-155 KO mice appeared to develop attenuated PH, with reduced levels of pro-inflammatory cytokines (TNF-a, IL-6). By integrating the gene expression profiling data from miR-155 KO mice with microRNA target predication, our data suggest that PPARα, an anti-inflammatory transcription factor, is a novel functional target of miR-155. Furthermore, we showed that in vivo silencing of miR-155 by miR-155 antagomir significantly ameliorated PH induced by Pristane. Consistently, the pro-inflammatory cytokines (TNF-a, IL-6) were reduced upon miR-155 silencing. Conclusion: Overall, our study shows that knockdown of miR-155 by miR-155 antagomir inhibits Pristane induced PH through reducing the production of pro-inflammatory cytokines. This finding suggests a promising therapeutic potential of miR-155 antagonist in the treatment of acute lung inflammation in lupus.
Role of OX40/OX40L signaling in the expansion of regulatory T cells in the NOD mouse model (BA13P.127)
Authors: Haddad Christine , Bhattacharya Palash , Prabhakar Bellur
Published: January 2023

Type 1 diabetes (T1D) is a T-cell-mediated disease characterized by destruction of pancreatic beta cells. Unfortunately, most treatment options for T1D are non-specific, with many side effects. Regulatory cells T (Tregs) play a crucial role in the maintenance of immune homeostasis against self-antigen. Many studies have revealed that Tregs in patients with various autoimmune diseases, including T1D, are diminished in number and/or dysfunctional. The therapeutic potential of Tregs has not been fully understood because generating large numbers of Tregs has remained a challenge. Recently, we reported that bone marrow precursor cells differentiated with GM-CSF (BMDCs) are able to selectively expand Tregs ex-vivo. Further investigation revealed that this expansion is TCR-independent but requires OX40/OX40L interaction. Thus, here we wanted to study whether treatment with soluble OX40L can expand Tregs in-vivo and in the NOD mice. Surprisingly, treatment with OX40L caused rapid onset of T1D in 12 wk but not 6 wk old mice. Analysis of spleens and lymph nodes showed an increase in Tregs only in the 6 wk old OX40L group. Thymic analysis revealed an increase in Tregs in both the 6 and 12 wk old OX40L groups. Interestingly, OX40L specifically induced the stable Tregs subset in 6 wk old mice, but the labile Treg subset in the 12 wk old mice. Based on these findings, we propose that OX40L can specifically induce different subsets of Tregs based on the cytokine milieu.
γδ T cells protect TCRα deficient mice from the autoimmunity induced by scurfy lymphocytes (BA13P.118)
Authors: Ujiie Hideyuki , Shevach Ethan
Published: January 2023

Foxp3+ regulatory T cells (Treg) are critical for the maintenance of immunological tolerance, and a mutation of the Foxp3 gene leads to the absence of Treg and a fatal systemic autoimmune disease in Scurfy mice. Transfer of Scurfy lymphocytes to RAG-/- recipients reproduces the inflammatory phenotype of the Scurfy donor including hepatitis and pneumonitis. Here, we show that TCRα-/- recipients, that lack TCRαβ+ T cells, but have TCRγδ+ T cells and B cells, are significantly protected from autoimmunity induced by adoptive transfer of Scurfy lymphocytes. Co-transfer of Scurfy lymphocytes and B cells from TCRα-/- mice into RAG-/- recipients failed to prevent inflammation. Furthermore, transfer of Scurfy lymphocytes to TCRβδ-/- mice, which lack both TCRαβ+ and TCRγδ+ T cells, but have B cells, resulted in more severe inflammation in the liver and lungs than that seen in TCRα-/- recipients. Taken together, these studies demonstrate that TCRγδ+ T cells, but not B cells, in the TCRαβ-/- recipients mediate protection from disease induced by Scurfy T cells. The TCRγδ+ T cells in the TCRα-/- recipients of Scurfy cells markedly expanded and expressed a highly activated (CD44hi CD62Llo) phenotype, but did not produce IL-10. Surprisingly, the Scurfy T cells in these recipients produced more IL-10 and less IL-4 than Scurfy T cells transferred to RAG-/- or TCRβδ-/- recipients, suggesting that TCRγδ+ T cells may protect TCRα-/- recipients by modulating cytokine production by Scurfy T cells.
Aberrant production of IL-13 by T cells promotes exocrinopathy in Id3 knockout mice (BA14P.206)
Authors: Belle Ian , Mahlios Joshua , Zhuang Yuan
Published: January 2023

Elevated cytokine IL-13 in serum has been associated with several autoimmune diseases, including Sjögren’s Syndrome. However, whether IL-13 plays an early and causative role in disease development is not known and cannot be easily studied in humans. Our previous work has shown that levels of the IL-13 are elevated in Id3 knockout mice, which has been established as a model for primary Sjögren’s Syndrome. Here, we utilized an IL-13 reporter to determine the source of the elevated IL-13 levels observed in Id3 knockout mice and assess its contribution to SS symptoms. Our results indicate that T cells, primarily CD4+ αβ T cells and γδ T cells, in Id3 knockout mice produce IL-13 at an elevated frequency well before disease symptoms become apparent. We also show that T cells developing early in life are more predisposed to produce IL-13. Finally, analysis of Id3 and IL-13 double deficient mice demonstrated that IL-13 plays an essential role in the deterioration of gland function. Our study indicates that enhanced IL-13 production by T cells is capable of inducing exocrinopathy in animal models.
IgA but not IgG serum antibodies to maize prolamins in Mexican patients with celiac disease (CD) and irritable bowel syndrome (IBS) (BA15P.226)
Authors: Sánchez Vargas Luis , Remes Troche Jose , Mellado Sánchez Gabriela , Vivanco Cid Héctor
Published: January 2023

High prevalence of CD has been found in IBS patients. CD is a chronic immune-mediated enteropathy triggered by exposure to dietary gluten in genetically predisposed individuals. Treatment for CD is to follow, a gluten free diet (GFD). However, some CD patients describe persistent symptoms despite the GFD, suggesting a cross reactivity or specific immune responses against other related-prolamins such as zein present in maize. We evaluated the prevalence of IgA and IgG anti-zein antibodies (AZA) in 37 CD, 375 IBS and 310 healthy controls (HC). All of the IBS subjects met the Rome III criteria and were negative to IgA h-tTG, IgA and IgG Gliadin II. IgA and IgG AZA were measured using an in-house ELISA. Elevated levels of IgA AZA were found in CD patients compared with IBS (p<0.001) and HC (p<0.01). CD patients had the highest prevalence (29.7%) compared with IBS (3.5%) and HC (1.3%) (p<0.0001). IgG AZA production was not demonstrated in any patients or HC. To explore if the results in IBS patients and HC can be explained by cross-reaction among gliadins and zeins, positive serum sample to IgA AZA were analyzed for IgA and IgG antibodies against native Gliadin (AGA). Interestingly, 53.8% of IBS patients and 50.0% of HC got negative results for IgA and IgG AGA, suggesting that IgA AZA response could be specific to maize prolamins. These results open the possibility to explore more about the contribution of the humoral responses to zein in the immunopathogenesis to CD and IBS.
The development of humanized mouse models to study immune regulation of autoreactive T cells. (BA15P.229)
Authors: Aryee Ken-Edwin , Brehm Michael
Published: January 2023

Type 1 diabetes (T1D) is the immune mediated destruction of insulin-producing beta cells in the pancreas, characterized by the loss of peripheral tolerance mechanisms needed to prevent autoimmunity. Regulatory T-cells (TREG) are a specialized CD4 T cell subset important for maintaining peripheral tolerance. IL-2 is critical to maintain TREG fitness and function, and deficiencies in the IL-2 receptor and signaling pathways contribute to the development of T1D. The lack of an in vivo system that fully recapitulates human T1D development impairs the ability to study the role of IL-2 signaling in T1D. Here we show that the Hu-PBL-SCID humanized mouse model can be used to study effector T cells and TREG from T1D-individuals. For these studies we injected PBMC from T1D or non-T1D donors into NOD Rag1null IL2rγnull (NRG) mice and followed the engraftment of human T cell populations. Our results show that PBMC from T1D donors engraft NRG mice with similar kinetics as compared to non-T1D donors. Moreover, our preliminary data show that expression of human IL-2 in recipient NRG mice improves T1D TREG frequency and survival. These results indicate that humanized mice are an effective model for studying autoreactive T cells from diabetic individuals
Effects of Rho GTPase CDC42 on Th2 and Th17 differentiation (CCR6P.277)
Authors: Yang Jun-Qi , Guo Fukun , Zheng Yi
Published: January 2023

We have reported that cell-division-cycle 42 (CDC42) of Rho GTPase family was important for T cell development and homeostasis with the use of conditional CDC42-deficient mice (CDC42flox/LckCre), in which CDC42 was deleted in CD4-CD8- double negative stage of thymocytes. Recently, we generated another line of conditional CDC42-deficient mice with selective expression of the Cre recombinase beginning at a late stage of T cell development (after positive selection of CD4+CD8+) using the distal promoter of the Lck (dLck) gene. We found that CDC42-deficiency (CDC42flox/dLckCre) did not affect T cell development, as expected. The development of natural regulatory T cells (nTreg) was also not affected by CDC42-deficiency. However, CDC42-deficiency impaired in vitro activation of both CD4+ and CD8+ T cells. When naïve CD4+ T cells were cultured under non-polarized conditions, CDC42-deficiency significantly reduced Th1 and Th2 cytokine secretion, but slightly increased Th17 cytokine secretion from CD4+ T cells. Under Th1/Th2/Th17-polarized conditions, CDC42-deficiency significantly downregulated Th2-differentiation, but was less effective to Th1 and Th17 differentiation. CDC42 has no significant role on the induction of Treg in vitro. The ongoing studies are trying to explore the underlying molecular mechanisms. Our findings suggest that CDC42 of Rho GTPase is not only crucial for T cell development and activation, but also involved in Th differentiation, especially Th2 cells.