bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance of diabetes in aged NOD mice is mediated by CD4+CD25+Foxp3+ regulatory T cells (P4144)
Authors: Guo Zhiguang , Liu Bolin , Tian Lei , Tian Bole , Ansarullah A. , Lu Yan , Holstein Martha , Deruyter Brittany
Published: January 2023

We investigated the role of CD4+CD25+FoxP3+ regulatory T cells on diabetes resistance in aged NOD mice that do not develop diabetes in pathogen-free housing. At 60 weeks age, <80% NOD mice developed diabetes. Adoptive transfer of lymphocytes from diabetic mice to aged nodiabetic NOD mice did not significantly increase the onset of diabetes. When 1x107 lymphocytes from age NOD mice were co-transferred with 5x106 lymphocytes from diabetes NOD mice into NOD.scid mice, all mice developed diabetes at 6 weeks. When 2x107 lymphocytes from aged NOD mice were co-transferred, none developed diabetes at 6 weeks and only 50% NOD.scid mice developed diabetes at 12 weeks. When 1x106 CD4+CD25- T cells was cotransferred with 5x106 lymphocytes from diabetic NOD mice, diabetes was developed in all NOD.scid mice. When 1x106 CD4+CD25+ T cells were used, only 17% mice developed diabetes at 12 weeks. The percentages of CD4+CD25+Foxp3+ T cells in the pancreatic lymph nodes, spleen and peripheral blood were 20.1±5.3%, 14.6±4.1% and 9.8±3.5% in aged NOD mice; 5.8±1.5%, 8.6±0.7% and 6.4±1.7% in control diabetic NOD mice. When a low dose of cyclosphamiade was given, diabetes was developed in 75% young NOD mice and in 18% aged NOD mice. However, when the high dose was given, 58% aged NOD mice had diabetes and CD4+CD25+Foxp3+ T cells in pancreatic lymph nodes were 7.0±1.7%. Our data demonstrated that immunoregulation through CD4+CD25+Foxp3+ T cells mediated the resistance of diabetes in aged NOD mice.
Immunogenic biologics: validation of screening, deimmunization and tolerization approaches (P3251)
Authors: Cousens Leslie , Moise Leonard , Terry Frances , Martin William , De Groot Anne
Published: January 2023

Immune responses directed against protein therapeutics can directly impact drug pharmacology, safety and efficacy. While many factors contribute to protein immunogenicity, T cell-dependent responses play a critical role. Tools to predict and reduce T cell responses to protein therapeutics present benefits at every stage of drug development. We provide evidence for two different approaches to mitigate therapeutic protein immunogenicity: epitope modification and antigen-specific tolerance induction. Following a systematic process of in silico epitope mapping applied to either Factor VIII or botulinum toxin, rational epitope modification resulted in reduced immunogenicity. In vitro and in vivo validation of computational predictions and iterative in silico modification of immunogenic epitopes were experimentally validated for selected modified sequences. Tolerance induction is achieved by co-delivery or by chemical or recombinant linkage of regulatory T cell epitopes (Tregitopes) to therapeutic proteins. We have demonstrated that Tregitope incorporation leads to lower immune responses against target epitopes identified in insulin, Factor VIII and botulinum toxin. Analysis, prediction, and modification of T cell-mediated immunogenicity of therapeutic proteins expands the available strategies for mitigating therapeutic protein immunogenicity, enables quality by design for drugs in development and improvement of licensed biologics to create biobetters.
Biological evaluation of human PBMC and Th17 cells using MILLIPLEX® MAP cell sgnaling and cytokine panels (P3255)
Authors: Wiese Rick , Warmke Tim , Keith Robert , Hwang Joseph
Published: January 2023

Peripheral Blood Mononuclear Cells (PBMC) are composed of all blood cells having a round nucleus. This includes lymphocytes, monocytes, and macrophages. The lymphocyte population is made up of T cells, B cells, and Natural Killer cells. Th17 cells are recently discovered T helper cell subtype that plays an important role in the establishment and maximization of the capabilities of the immune system. Th17 cells are derived from CD4+ lymphocytes which are prevalent in human PBMC samples. In this study, we used several multiplex cell signaling immunoassay panels and a human Th17 cytokine multiplex immunoassay panel to compose a biochemical profile of PBMCs before, during, and after a Th17 differentiation process. We also examined the effect of serum starvation and refeeding on undifferentiated and differentiated PBMC. Our differentiation process consisted of a 7-day treatment of four cytokines, IL-1β, IL-6, IL-23, and TGFβ. Cell signaling panel results demonstrated that several analytes exhibited increased constitutive expression and phosphorylation after differentiation and stimulation with serum. Additionally, the human Th17 cytokine panel detected a significantly increased secretion of many cytokines including Th17 cell specific cytokines from human PBMCs (e.g. IL-17F). In conclusion, the Th17 and cell signaling panels will be useful tools for Th17 cell-related cytokine and signaling profiling in PBMC and various biological samples.
Detection of microparticles in human plasma using flow cytometry: limitations and optimization techniques. (P3265)
Authors: Norris Philip , Danesh Ali , Hirschkorn Dale , Inglis Heather
Published: January 2023

Microparticles (MPs) range in size from 40 nm to 1 um. Flow cytometry (FCM) is the most commonly used method for analyzing MPs; however, accurate characterization of MPs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques for overcoming these limitations. To reduce background, we decreased antibody concentration, increased sample dilution, and reduced sample flow rate. Antibodies (Abs) to CD14, CD16, CD19, and CD235a showed high levels of aggregation resulting in false positive events in the MP FSC/SSC gate, while Abs to CD28, CD62L, and CD41a did not have significant aggregates. Switching Ab manufacturers was effective only at resolving background for one Ab, CD235. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-MP events. We show that our antibody filtration method removes aggregates without compromising the effectiveness of the dye to identify/label antigen-expressing cells. Furthermore, we found our pre-filtration method to be slightly more effective at removing aggregates than a centrifugation method described in the literature. In conclusion, use of these optimized techniques enhances the accuracy and reliability of MP detection using FCM.
Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error (P3233)
Authors: Chan Leo , Laverty Daniel , Smith Tim , Nejad Parham , Hei Hillary , Gandhi Roopali , Kuksin Dmitry , Qiu Jean
Published: January 2023

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method.
Delivery of vaccine genes and proteins into dendritic cells using the bacteriophage T4 DNA packaging machine (P3273)
Authors: Rao Venigalla , Tao Pan , Mahalingam Marthandan , Bernard Marasa , Zhang Zhihong , Chopra Ashok
Published: January 2023

Traditionally, vaccines are produced by empirical methods and current platforms generally do not allow complex designs to modify the quality and quantity of immune responses. We have developed a bacteriophage T4 platform that allows construction and delivery of multivalent vaccines containing both DNA and protein. Using this system, the composition of vaccines can be customized to address mechanistic questions as well as develop efficacious vaccines. The phage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56µm-long 170-kb viral genome into 120nm x 86nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, Hoc and Soc. T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells, or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein elicited robust antibody and cellular immune responses. This “progene delivery” approach might lead to new types of genetic therapies and prime-boost vaccines against complex infectious diseases such as HIV and malaria.
Methods for defining vascular and parenchymal leukocyte populations: implications for characterizing innate and adaptive immune responses in non-lymphoid tissue (P3281)
Authors: Anderson Kristin , Sung Heungsup , Mayer-Barber Katrin , Beura Lalit , Vezys Vaiva , Barber Daniel , Masopust David
Published: January 2023

Defining the anatomic location of cells of the innate and adaptive immune system is critical for understanding immune responses to infection. Recently, we published that intravascular staining can distinguish CD8 T cells located in the lung tissue from those confined to capillary vasculature. Remarkably, up to 95% of CD8 T cells isolated from lung were present within capillaries, despite extensive perfusion, and removing these cells from analysis resulted in revised interpretations of lung T cell homing and differentiation. We now show that perfusion results in a disruption of lung architecture and a loss of parenchymal cells of interest. Moreover, we expand our analysis to other leukocyte populations (lymphoid and myeloid) in additional non-lymphoid tissues and in the context of a variety of disease models, including graft versus host disease and infection with influenza or Mycobacterium tuberculosis. We have defined revised methodologies that, if applied, will have a pronounced impact on the interpretation of studies examining numerous leukocyte subsets in many non-lymphoid compartments. We are now utilizing these methods to examine the dynamics of primary and anamnestic responses to, and mechanisms of protection against, respiratory infections.
Dissecting IL-22 biology using a fate reporter mouse (P6263)
Authors: Ahlfors Helena , Li Ying , Biro Judit , Potocnik Alexandre , Stockinger Brigitta
Published: January 2023

Interleukin-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A number of IL-22 producing cell types has been identified, but questions remain regarding their stability and relationship to other subsets. We therefore generated a fate reporter mouse that would allow not only the identification of IL-22 producing cells but also their fate mapping in vivo. To trace cells expressing IL-22, a sequence encoding Cre recombinase was cloned into the Il22 locus (Il22Cre). To visualize Cre activity, the IL22Cre mice were then crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under the control of the endogenous Rosa26 promoter (R26ReYFP). In IL22CreR26ReYFP mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression regardless of the present production status of this cytokine. Using this mouse strain we show that IL-22 expression is restricted to immune cells, the main producers being innate lymphoid cells in gut and γδT cells in skin or lung in an unchallenged mouse. In a model of imiquimod induced psoriasiform skin inflammation we observed a substantial accumulation of IL22-eYFP+ cells, demonstrating the functionality of our reporter in vivo. Further studies are aimed at investigating the role of IL-22-producing cells in infectious, inflammatory and autoimmune diseases and to delineate their relationship with other cytokine producing cell subsets, notably innate lymphoid cells.
Accelerated vitiligo is associated with enhanced IL17 responses in mice (P5166)
Authors: Kang Hee-Kap , Eby Jonathan , Klarquist Jared , Mehrotra Shikhar , Nishimura Michael , Jack Longley B. , Caroline Le Poole I.
Published: January 2023

Vitiligo is a pigmentary skin disease resulting from T cell mediated cytotoxicity towards melanocytes. To study the involvement of Th17 cells in vitiligo pathogenesis, we generated a spontaneous mouse model of epidermal depigmentation in vitiligo by crossing HLA-A2 transgenic mice expressing a human-derived, tyrosinase reactive T cell receptor transgene within T cells were crossed with a K14-SCF transgenic mouse that retains intra-epidermal melanocytes. In this triple (HLA-A2, h3T, K14-SCF) transgenic mouse, we observed that hair depigmentation was significantly accelerated as compared to the h3TA2 mice parental strain. The presence of the SCF transgene is associated with an increase of IL-17A+T cells among peripheral T cells. Triple transgenic display enhanced tyrosinase-specific Th17 responses and stronger spontaneous CTL responses. The increased Th17 responses are correlated to expansion of SCF responsive MM cell for megakaryocyte and/or bipotent megakaryocyte/erythroid progenitors (CD117+CD151+CD41+CD11c-CD11b-) that induce Th17 cells as previously shown to mediate lupus. In addition, SCF overexpression allows for spotted repigmentation of the epidermis to form ‘NeVi’. Repigmentation is accompanied by reduced Th17 and increased Treg infiltration to the skin. Taken together, accelerated vitiligo is associated with IL-17 responses in mice.
Human regulatory T cells are hyper-responsive to low levels of IL-2 (P5156)
Authors: Malek Thomas , Yu Axin , Vendrame Francesco , Snowhite Isaac , Pugliese Alberto
Published: January 2023

Our recent work demonstrates that weak IL-2R signaling supports the development and homeostasis of mouse Treg cells. This finding provides a conceptual framework for the use of low dose IL-2 in immunotherapy to selectively promote Treg cells for tolerance induction. To begin to examine the extent that low dose IL-2 may selectively activate human Treg cells, we assessed IL-2-dependent tyrosine-phosphorylation of STAT5 (pSTAT5) in various populations of IL-2R-bearing human lymphocytes. Dose-response studies indicate that Treg cells are 50-100 fold more responsive to IL-2 for pSTAT5 activation than CD4 T memory and T effector (Teff) cells. Relative high levels of high affinity IL-2Rs on Treg cells provide a partial explanation for this enhanced signaling sensitivity to IL-2. However, other factors are also clearly involved in this response because in vitro-derived CD4 Teff cells express much higher levels of CD25, CD122, and CD132 than Treg cells, but these Teff cells remain much less responsive to IL-2 when compared to Treg cells. This finding indicates that human Treg cells utilize a cell intrinsic mechanism to amplify IL-2R signaling when IL-2 is limiting. Overall these studies provide an explanation for the apparent safety of low dose IL-2 therapy for patients with autoimmune conditions where Treg cells were enhanced without re-activation of autoreactive T cells.
Increased BAFF concentrations in patients with dilated cardiomyopathy and impact on response to immunotherapy (P4548)
Authors: Alahmad Adnan , Eckerle Lars , Dörr Marcus , Riad Alexander , Felix Stephan , Hundt Matthias
Published: January 2023

DCM is a heart disease characterized by ventricular chamber enlargement and systolic dysfunction. Autoantibodies against cardiac proteins have been described in DCM. Removing these IgG antibodies from the blood by immunoadsorption with subsequent IgG substitution (IA/IgG therapy) can improve the clinical outcome and heart function. To determine whether the increased number and survival of autoreactive B cells in DCM patients could be related to the overexpression of BAFF, we measured BAFF in the plasma of groups of 59 DCM patients and 37 age-matched controls by ELISA. Thereafter, DCM patients were treated with IA/IgG therapy, and left ventricular ejection fraction (LVEF) was measured directly after therapy and after 6 months to quantify heart function. BAFF was significantly elevated in DCM patients compared to controls [2333±124 pg/ml vs. 927±58 pg/ml (mean±SEM), p<0.0001]. Similar results were obtained for BAFF mRNA expression in PBMC. After IA/IgG therapy, DCM patients were divided into 2 groups according to their change in LVEF: increase of ≥20% (responder), and decrease or increase of <20% (non-responder). BAFF in the non-responder group was slightly, but significantly higher than in the responder group (3107±357 pg/ml vs. 2078±151 pg/ml, p<0.01). These results suggest that BAFF might play an important role in the pathophysiology of DCM. Moreover, patients with the highest levels of BAFF might represent a subgroup, which does not respond well to IA/IgG therapy.
Antinuclear antibodies in general population: what does that mean? (P4535)
Authors: Raj Prithvi , Li Quan-Zhen , Karp David , Olsen Nancy , Sivils Kathy , James Judith , Kelly Jennifer , Lauwerys Bernard , Gregersen Peter , Wakeland Edward
Published: January 2023

Antinuclear antibodies (ANAs), the serologic hallmarks of autoimmunity are frequently seen in patients with systemic autoimmune diseases. We screened 2223 Caucasian normal control and 143 SLE sera for ANA, anti-chromatin, anti-Ro and anti-SSB antibodies. Further, we employed protein array screening to detect antibodies against over 90 different auto-antigens. Our data show that about 26% of the normal population are ANA positive, with 17% (382 of 2223) having moderate (20-40 ELISA units) and 9% (197 of 2223) having high (>40 ELISA units) ANA antibody levels. Chromatin ELISA and Crithidia luciliae immunofluorescence tests (CLIFT) confirmed the absence of anti-chromatin antibodies in the ANA positive fraction of the normal population. Results of Anti-Ro-52 ELISA showed that 6.5% (3/46) moderate ANA NCs, 40.4% (17/42) high ANA NCs and 26.3% (25/95) SLEs test positive for anti-Ro-52 antibodies. Interestingly, a very strong correlation was observed between ANA and anti-Ro-52 antibodies in the high ANA (Pearson r =0.69, p<0.0001) and SLE group (Pearson r =0.66, p<0.0001). We also investigated ANA positivity as a function of age in the normal population and found that correlation between ANA and Ro-52 antibodies was stronger in subjects under 50 year of age (Pearson r =0.75, p<0.0001) than that of those above 50 year (Pearson r =0.57, p<0.0001). This observation was also consistent in SLE patients (Pearson r = 0.76, p<0.0001). Protein array results will be discussed.
Autoreactive T cell activation is sufficient for breaking B cell tolerance in the setting of functional antigen-specific dominant tolerance (P4543)
Authors: Bumpus Tia , Marshak-Rothstein Ann
Published: January 2023

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by anti-nuclear autoantibody (ANA) production by activated autoreactive B cells and the persistence and activation of autoreactive T cells. Animal models of lupus provide evidence that T and B cells interact to drive autoimmunity. Whether a break in tolerance in the T cell or B cell compartment could lead to a break in tolerance in the other compartment is unclear. Several studies have addressed this issue using transgenic autoreactive B cells, but whether significant elaboration of autoreactive B cells in the normal repertoire can be driven by activated T cells has not been shown. To address this question, we developed a mouse model in which naïve or activated transgenic autoreactive CD4+ T cells are transferred into hosts expressing their cognate pseudo-autoantigen (autoAg) on B cells and other antigen presenting cells (APCs). Interestingly, autoAg-specific Th1 or Th2 cells, but not naïve cells, drive ANA production by endogenous autoreactive B cells. In bone marrow chimeras, autoAg expression by radioresistant APCs was crucial for sufficient suppression of B cell activation by activated T cells. Additonally, we found that radioresistant APCs most effectively activate autoAg-specific splenic Tregs. We have shown that T cell activation is an important checkpoint in the prevention of ANA production by autoreactive B cells and that antigen-specific Tregs most effectively control ANA production.
Heme oxygenase-1 expression prevents inflammation in acute cardiac injury (P5190)
Authors: Hull Travis , Bolisetty Subhashini , Dealmeida Angela , Litovsky Silvio , Prabhu Sumanth , Agarwal Anupam , George James
Published: January 2023

Heme oxygenase-1 (HO-1) degrades pro-oxidant heme into iron, carbon monoxide, and biliverdin. Overexpression of HO-1 prevents chronic heart failure by modulating the inflammatory response. However, when HO-1 must be induced in the course of injury to prevent cardiac failure has not been determined. We hypothesized that induction of HO-1 overexpression at the onset of acute cardiac injury prevents cardiac dysfunction through modulation of the innate immune response. We generated mice with inducible overexpression of a human HO-1 (hHO-1) transgene (MHC-HO-1 mice) by breeding mice with cardiac-specific expression of a tamoxifen (TAM)-inducible Cre recombinase (MHC-Cre mice) with mice containing an hHO-1 transgene preceded by a floxed stop signal (CBA-flox mice). Maximal induction of hHO-1 occurs 3 days after TAM administration (40 mg/kg body weight on two consecutive days) in MHC-HO-1 mice. In accord with previous reports, we found that MHC-Cre mice develop acute systolic dysfunction after TAM administration, which is characterized by severe inflammation, diffuse cardiomyocyte necrosis, and >80% mortality. There was an 18-fold increase in cardiac neutrophils in MHC-Cre mice treated with TAM, but the mononuclear phagocyte population was unchanged. In MHC-HO-1 mice, cardiac hHO-1 overexpression attenuated acute cardiac injury, as mortality and systolic dysfunction caused by Cre recombinase were prevented and there was no evidence of myocyte damage or cardiac inflammation.
LMP1 signaling in vivo downregulates DUSP1 and exacerbates inflammatory responses (P5204)
Authors: Stunz Laura , LeBeau Robert , Bishop Gail
Published: January 2023

The EBV oncogenic protein LMP1 is implicated in pathogenesis of B cell malignancies and exacerbation of autoimmune disease. We previously produced mice that express, in B and myeloid cells, a transgene with the external domain of CD40 and signaling domain of LMP1. CD40-LMP1 substitutes for CD40 to induce a humoral response in CD40-/- mice, but they display elevated autoantibodies, enlarged lymphoid organs, and elevated inflammatory cytokines. Co-expression of CD40 and CD40-LMP1 partially ameliorates this inflammatory/autoreactive phenotype. The phosphatase DUSP1 is downregulated in B cells from CD40-LMP1tg mice, and upon induced expression of Wt LMP1 in a B cell line. DUSP1 is implicated in the control of inflammatory signaling pathways, and DUSP1 deficiency has been shown to increase susceptibility of mice to LPS induced sepsis. We hypothesized that inhibition of DUSP1 expression caused by LMP1 signaling contributes to the association of LMP1 with inflammation. We tested the impact of LMP1 expression on in vivo responses to LPS in mice expressing CD40-LMP1, CD40, or both. Two hrs after challenge with a low dose of LPS, serum TNFa and IL1b were increased to a markedly greater degree in mice expressing CD40-LMP1, and this was alleviated by co-expression of CD40. Additionally, >50% of CD40-LMP1 Tg mice, but none of the mice expressing Wt CD40, developed sepsis. These findings suggest a potential mechanism by which LMP1 promotes exacerbation of chronic inflammatory disease.
Comparison of pharmacokinetics and biological activity of two IL-15 heterodimeric forms in mice and macaques (P6356)
Authors: Bergamaschi Cristina , Valentin Antonio , Kulkarni Viraj , Bear Jenifer , Rosati Margherita , Alicea Candido , Sowder Raymond , Chertova Elena , Lifson Jeffrey , Felber Barbara , Pavlakis George
Published: January 2023

IL-15 is a member of the γ-chain family of cytokine important for the proliferation and function of NK and T cells. IL-15 acts on the surface of the cell in complex with IL-15Rα to engage IL-15Rβγ complex in nearby cells. We recently showed that in both murine and human sera IL-15 is in complex with soluble IL-15Rα (sIL-15Rα), suggesting that IL-15/IL-15Rα heterodimer is responsible for IL-15 bioactivity in vivo. We have generated stable cell lines secreting high levels of fully processed human IL-15/sIL-15Rα cytokine. We also prepared an additional form of human glycosylated IL-15 heterodimer in which the C-terminus of sIL-15Rα is fused to the Fc region of human IgG1. The biochemical properties and bioactivity of these IL-15 heterodimers were tested in mice and macaques. IL-15/IL-15RαFc was characterized by an increased bioactivity that reflected its superior plasma half-life in mice, in comparison to IL-15/sIL-15Rα. However, delivery of these two IL-15 heterodimers in macaques failed to reveal any major differences in their pharmacokinetic and pharmacodynamic profile. In macaques, both IL-15 heterodimeric forms resulted in similar levels of plasma IL-15 and induced similar proliferation of NK, γδ and CD8+ T cells. Also, both IL-15 heterodimers were effective in reducing tumor burden in a colon carcinoma mouse model. These results provide important insights for the choice of IL-15 for clinical use, and suggest caution when interpreting data using IL-15/IL-15RαFc in mice.
Finding new receptors for serum amyloid A (P6362)
Authors: He Rong , Omer Linda
Published: January 2023

Serum amyloid A (SAA) is a major acute-phase protein with cytokine/chemokine-like properties. It can be expressed at sites of inflammation and induce the migration of immune cells to the inflammatory sites. SAA plasma concentration can be 1000 times higher in the patients with autoimmune diseases such rheumatoid arthritis, than in the healthy people. However, its precise role in inflammation and infection remains unclear. SAA is known to bind to several receptors such as FPRL1, TLR2, and TLR4, etc. It is possible that SAA also use other chemokine receptors to recruit immune cells during the inflammation. In this study, we used a mathematical approach called Natural Vector Method (NVM) and analyzed the genetic information of all major human chemokines (total 175) and SAA. SAA was found clustered with two chemokines based on both DNA and protein sequences: CXCL9 and CCL23. We hypothesized that SAA may use these chemokines’ receptors, CXCR3 and CCR1. We found that SAA indeed can induce MAP kinase ERK activation in the receptor over-expressed cell line. We will check other receptor-mediated cell functions such as intracellular calcium mobilization, to confirm that SAA can use these two receptors. We hope that our research work not only helps to understand the function of SAA, but also introduces a new way of finding receptors for the endogenous molecules.
KMT1E is a regulator of T cell development (P4542)
Authors: Martin Francis , Nicholson Thomas , Loureiro Joseph , Chen Taiping , Huang Qian
Published: January 2023

The histone methyltransferase KMT1E has a demonstrated role in pluripotency, embryonic stem cell development, and melanoma formation, yet its role in T cell development has not been investigated. As T-cell development involves many stages of lineage commitment, we hypothesized that KMT1E may have a role in regulating these commitment stages. KMT1E is dynamically regulated during the stages of T-cell development, peaking at the DN2 through DN4 stages and dramatically waning as lineage commitment occurs. In order to further query the role of KMT1E in thymocyte development, we created conditional knockout animals in which KMT1E is deleted specifically in the T cell compartment (where the Cre recombinase is driven by the Lck promoter). Deletion of KMT1E caused a profound reduction in the numbers of DN1 (Lin-/CD44+/CD25-) cells in the thymus and single positive CD4 and CD8 cells in the thymus and spleen. In order to elucidate the genetic changes induced by KMT1E that may influence the observed phenotype, we performed microarray experiments on thymocyte precursors from the wild type and conditional knockout animals. Deletion of KMT1E had a modest effect on global gene expression, with only a small subset of the genes being upregulated. Currently we are examining the role of these genes mediating KMT1E’s function in early T cell development.
The key role of Aquaporin 3 and Aquaporin 10 in the pathogenesis of pompholyx (P6372)
Authors: Soler David , Bai Xiaodong , Ortega Lina , Nedorost Susan , Cooper Kevin , McCormick Thomas
Published: January 2023

Pompholyx (a.k.a dyshidrotic eczema) is a skin affliction without a compelling pathophysiological trigger. The disease is characterized by the sudden onset of blisters exclusively in the hands and soles that can expand and fuse, eventually leading to heavily dehydrated skin. Using immunofluorescence we found that the aquaglyceroporins Aquaporin 3 (AQP3) and Aquaporin-10 (AQP10) are aberrantly expressed across the suprabasal keratinocytes in the involved palm lesions of pompholyx patients compared to uninvolved skin and skin from healthy controls. AQP3 and AQP10 function as bi-directional transport channels for water and glycerol, and their activity is inhibited at acidic pH and favored at basic pH. The suprabasal keratinocyte presence of these two aquaglyceroporins may bridge the abundantly hydrated dermis and basal epidermis to the outer environment in pompholyx, creating a dehydrating osmotic gradient imbalance as a result from exposure to exogenous water. Since hands are typically exposed to water numerous times daily, this constant osmotic gradient imbalance may provide a mechanistic explanation for the extreme dehydration observed clinically in pompholyx skin patients, thus irreversibly altering the skin homeostasis and contributing to the chronic cutaneous inflammation of this disease.
Cutaneous immuno-surveillance and regulation of inflammation by group 2 innate lymphoid cells (P6374)
Authors: Roediger Ben , Kyle Ryan , Yip Kwok , Sumaria Nital , Guy Thomas , Mitchell Andrew , Tay Szun , Jain Rohit , Forbes-Blom Elizabeth , Chen Xi , Tong Philip , Bolton Holly , Paul William , Fazekas de St. Groth Barbara , Grimbaldeston Michele , Le Gros Graham , Weninger Wolfgang
Published: January 2023

Type 2 immunity is critical for defense against cutaneous infections, but also underlies the development of allergic skin diseases. We report the identification in normal murine dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depends on IL-7 and constitutively produces IL-13. Intravital multiphoton microscopy revealed that dermal ILC2 specifically interact with mast cells, whose function is suppressed by IL-13. Treatment of Rag1−/− mice with IL-2 led to a marked expansion of activated, IL-5-producing dermal ILC2, resulting in the development of spontaneous dermatitis characterized by eosinophil infiltrate and activated mast cells. Our data uncover a novel interactive pathway between two innate immune cell populations implicated in type 2 immunity, and demonstrate that ILC2 exhibit both pro- and anti-inflammatory properties.
Development and evaluation of an in vitro skin sensitization assay based on primary monocyte-derived dendritic cells (P6376)
Authors: Kuehnl Jochen , Gerlach Silke , Reuter Hendrik , Onken Stefan , Wenck Horst , Schepky Andreas
Published: January 2023

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Until now, animal experiments (e.g. the local lymph node assay) are supplying most of the data used to assess the sensitization potential of new chemicals. However, becoming effective in March 2013, the 7th amendment to the EU Cosmetic Directive introduced an animal testing ban for the endpoint skin sensitisation for cosmetic ingredients. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. We successfully solved the issues associated with the use of primary cells (optimized cytokine concentrations, incubation time, readout, pooled vs. single donors and cytoxicity) resulting in a stable and reproducible protocol. Here, we report on the adaption of the protocol to the 96-well format, allowing for a higher and cost-effective throughput of test substances. Comparative analyses of the effect of several skin sensitizers on CD86 expression display similar results for 24- and 96-well format. Upscaling of the PBMDCs advances its applicability for the reliable detection of human skin sensitizers.
Asian sand dust induces autophagic markers and activates NF-κB in immune cells (P6377)
Authors: Song Yuan , Ichinose Taksmichi , Yoshida Yasuhiro
Published: January 2023

It is well going to know that several particles modulate immune responses and phagocytosis may induce activation in antigen-presenting cells. Asian sand dust (ASD), which is derived from air pollutants in East China that migrate to Japan and ASD is concerned about influence for health. Our group has reported that ASD induced inflammatory responses in lung tissue and causes respiratory diseases in mice. In the last meeting, we reported that the effect of ASD on the immune system in vivo. In this study, we examined the effect of ASD in vitro. Cell lysates were prepared from freshly isolated splenocytes of ICR mice and resolved by western blotting. Immunoblotting demonstrated that ASD induced autophagic-marker LC3-II expression and decreased the level of p62 especially in CD11b+ cells. Cell activation of splenocytes was evaluated by a cell viability assay. The cell viability assay showed mitogen-induced proliferation of splenocytes is inhibited by ASD treatment. In addition, an electrophoretic mobility shift assay demonstrated increased activated-NF-κB in bone marrow derived macrophage after ASD treatment. Taken together, these results suggest that ASD activates immune cells, and may modulate cell survival or activity.
Immune cell profiles in response to silver nanoparticles associated with medical devices (P3357)
Authors: Zelikoff Judith , Willis Daniel , Degheidy Heba , Zhang Qin , Umbreit Thomas , Goering Peter
Published: January 2023

Silver nanomaterials are increasingly being used as antimicrobial agents in medical devices, yet their effects on immunocompetence are unclear. This study investigated modulation of immune cell profiles and cell activation by exposure to silver nanoparticles (AgNP). Mice were injected iv (each day for 2 d) with 66 or 300 µg of either 20 or 110 nm AgNP (or sodium citrate) and sacrificed 1 d later. Flow cytometric analysis revealed that injection of 66 µg of 20 nm AgNP increased Th2 cells and decreased effector memory cells, while exposure to the same dose of larger-sized AgNP had no effect on numbers of CD3+,CD4+, Th1, Th2, Th17 cells, CD4/CD8 ratio, or total memory/naïve/central memory/effector memory cells compared to controls. At 300 µg, 20 nm AgNP significantly decreased the number of naïve CD4+ cells and CD3+CD4- cells (i.e., cytotoxic T cells), while increasing central memory cells, activated central memory cells, effector memory cells, activated effector memory cells, total memory cells, and natural killer cells. A somewhat different cell profile was observed following exposure to the same dose of 110 nm AgNP including an increase in activated CD11c+ cells (macrophages). Taken together, these data indicate that systemic AgNP exposure can modulate the immune system in a dose-dependent manner, with smaller size particles leading to more drastic effects. Overall, these studies have policy implications and demonstrate the need for protecting already vulnerable individuals.
Longitudinal tracking of immune cell subsets and phenotypes in cynomolgus monkeys following immunization with two model antigens (P3369)
Authors: Casey Kerry , Wang Shu , Gallagher Sandra , Stern Jessica , Xiong Ximing , Manetz Scott , Grant Ethan , Herbst Ronald , Carter Laura L. , Ettinger Rachel
Published: January 2023

Antigen immunization studies in cynomolgus monkeys (cyno) provide an opportunity to study in vivo immune responses in a higher order species. Such models can also be useful for testing candidate immunomodulatory drugs which lack rodent cross-reactivity. Here, we have optimized and validated eight, 6-7 color panels of cyno-human cross-reactive antibodies allowing extensive flow cytomtric phenotyping of immune cell populations under steady state, Tetatanus Toxoid (TT), or Keyhole limpet hemocyanin (KLH) primed conditions. Comparisons were made between fresh & overnight cells in Na/Hep tubes to cells phenotypically stabilized in Cytochex tubes. We followed 17 distinct lymphocyte subsets including T, B & NK cells and tracked their activation status over 85 days following primary or secondary immunization. Similar to humans, cyno B & T cell compartments demonstrated a history of prior stimulation evidenced by elevated memory:naive cell ratios and significant baseline expression of CD95, GrB, Ki-67, PD-1, CXCR3, CCR6 and CD11c. In response to immunization, frequencies of circulating Ki67+ B & T cells increased, followed by an elevation of T dependent Ab titers. Changes in Th1 and Th17 subset frequencies as well as CD25 and CD69 expression following immunization were also observed. Direct application of these Ab panels to leukocytes from Rheumatoid Arthritis patients has recapitulated published observations supporting the utility of modeling immune responses in cyno.
IgG 4-RSD involving the pleura (P3373)
Authors: Kohli Ritesh , Chowdhary Harjinder
Published: January 2023

IgG4-RSD is a novel clinical entity that shows IgG4 producing plasma cells in affected organs with fibrotic and sclerotic changes, and elevated serum IgG4 levels. IHerein, we report one case of IgG4-RLD, presenting with pleural effusion, with lab values showing high IgG4 levels and pleural biopsy showing infiltrated plasma cells with strong immunoreactivity for IgG4. A 55year old Caucasian female, nonsmoker with no significant past medical history presented to the clinic for b/l hand swelling and occasional dyspnea. Physical examination was unremarkable except for decreased breath sounds left lung, and b/l hand swelling.There was no evidence of synovitis involving hands or any other joints. Range of motion was normal. The routine blood tests Immunological markers were normal except high IgG-4levels Pleural fluid aspiration was suggestive of exudative effusion. Pleural biopsy was performed that was suggestive of massive lymphoplasma cell infiltration in the pleura, with immunostaining revealing predominant IgG4 positive plasma cells greater than 30hpf.He was started on steroid therapy with clinical significant improvement in few weeks. IgG4RSD is an unrecognized condition about which knowledge is now growing rapidly., Acknowledging that IgG4RSD may present as pleural effusion is essential for early management and preventing complications
Phospholipase D1 regulates the extent of TCR-induced lytic granule release by CD8+ CTL (P1390)
Authors: Radoja Sasa , Ma Jennifer
Published: January 2023

Regulation of lytic activity in CD8+ CTL remains incompletely understood despite the importance of this effector T cell function in immune response to intracellular pathogens and tumors. One important aspect involving the regulation of T cell effector function is the spatio-temporal regulation of DAG metabolism in T cells. Based on the involvement of phospholipase D (PLD) isoforms in DAG metabolism-related regulation of vesicular trafficking in other cells types, we reasoned that these enzymes might regulate lytic function in CD8+ T cells. By combining the use of pharmacological inhibitors and siRNA-mediated knockdown, we showed that PLD1 isoform modulates lytic function in primary mouse CD8+ CTL. Surprisingly, PLD1 knockdown in CTL lead to the enhancement of lytic activity despite its inhibitory effect on conjugate formation. The observed phenotype was not due to increased levels of expression of lytic molecules but was due to an increased release of lytic granule contents in response to TCR engagement. At the same time, overexpression of PLD1 in CTL resulted in the inhibition of lytic activity that coincided with an accumulation at the immunological synapse of both phosphatidic acid (PA), a byproduct of PLD1, and actin. Thus, PLD1 regulates the degree of lytic response in CD8+ CTL, potentially via PA-mediated induction of actin polymerization at the synapse, which could serve as a molecular gate that controls the extent of lytic granule release toward target cells.
Vitamin D3 is necessary for human dendritic cells to respond to bacterial products (P1372)
Authors: Brosbol-Ravnborg Anne , Bundgaard Bettina , Höllsberg Per
Published: January 2023

Background: Differentiated DCs are matured in the presence of lipopolysaccharide, a product of gram-negative bacteria. We hypothesized that additional bacterial products are able to cause DC maturation and that 1,25-dihydroxyvitamin D3 is capable of regulating this process by controlling cytokine secretion. Methods: DCs were differentiated and matured from human monocytes in the presence or absence of vitD3. DC maturation was induced by lipopolysaccharide and peptidoglycan. DC differentiation and maturation were monitored by morphology change and expression of surface molecules. Cytokine secretion of IL-6, IL-8, IL-10, and IL-12p70 was measured by ELISA. Expression of vitD3 receptor in DCs was determined by Western blotting. Results: The presence of vitD3 inhibited LPS-induced DC maturation, including formation of dendrites on DCs consistent with the phenotype of immature DCs. VitD3 was necessary for LPS- and PGN-induced secretion of IL-6, IL-8 and IL-10, whereas vitD3 inhibited LPS-induced secretion of IL-12. Furthermore, both LPS and PGN could act in synergy with vitD3 to increase the expression of the vitD3 receptor in DC. Conclusions: The presence of vitD3 was necessary for LPS- and PGN-induced secretion of IL-6, IL-8 and IL-10 in DCs. The expression of vitD3 receptor in LPS- or PGN-matured DC was enhanced in a synergy with vitD3. Elucidating the mechanisms behind this may be of importance for understanding the role of vitD3 during induction of peripheral tolerance.
Retinoic acid-rich microenvironment provides clonal survival cues for tumor and bacteria-specific CD8+ T cells (P1376)
Authors: Guo Yanxia , Pino-Lagos Karina , Noelle Randolph
Published: January 2023

While studies have implicated a role of vitamin A (VA) in host resistance to infectious disease, little is known as to the role of VA and its active metabolite, retinoic acid (RA) in host defense against cancer. Herein, it is shown for the first time that local RA production within the tumor microenvironment (TME) is increased up to 5-fold compared to naïve surrounding tissue, with a commensurate increase in RA signaling to regionally infiltrating tumor-reactive T cells. Conditional disruption of RA signaling in CD8+ T cells using a dominant negative Retinoic Acid Receptor α (dnRARα) illustrated that RA signaling is required for tumor-specific CD8+ T cell expansion/accumulation and protective anti-tumor immunity. Specific deletion of RARα, RARβ and RARγ in CD8+ T cells further demonstrated that RARα is required for CD8+ T cell survival, whereas RARβ is indispensable for bacteria-specific CD8+ T cell expansion in response to bacterial infection. These studies established a novel function of RA essential for both anti-tumor and infection CD8+ T cell immunity.
Inhibition of acetylcholinesterase regulates the anti-microbial immune response (P1378)
Authors: Fernandez-Cabezudo Maria , Al-Barazie Ray , al-Ramadi Basel
Published: January 2023

We have previously demonstrated that inhibition of the enzyme acetylcholinesterase, leads to an enhancement of animal survival following an oral infection with a lethal strain of S. typhimurium, a Gram-negative, facultative, intracellular pathogen. We hypothesized that the resultant increase in acetylcholine levels modulates the macrophage inflammatory response to infection. In this study, we assessed the potential of modulating the host’s immune response following a lethal bacterial infection. BALB/c mice were orally infected with a virulent strain of S. typhimurium, following which they were injected i.p. with the acetylcholinesterase inhibitor paraxon. The start of paraoxon injection varied from 30 minutes to 24 hrs post bacterial infection and repeated daily for 7 consecutive days. In contrast to control group which exhibited 10% survival, animals treated with paraoxon up to 6 hrs post infection showed 50% overall survival. Enhanced survival observed in this group correlated with a more robust inhibition of bacterial proliferation in systemic target organs. Furthermore, preliminary evidence from flowcytometric analysis of mesenteric lymph nodes and spleen cell populations revealed an increase in T lymphocyte ratios as well as upregulation of IFN gamma-regulated activation markers on B and T lymphocytes in the paraoxon-treated group. These findings underscore the importance of the neural-immune axis in regulating immunity to bacterial infection.
Expression of interferon-inducible genes is influenced by sex hormones and gender in SLE (P1380)
Authors: Hossain Awlad , Hahn Bevra , Singh Ram
Published: January 2023

Women may be more susceptible than men to SLE and other autoimmune diseases in part because many healthy women have higher interferon regulated genes and fewer regulatory T cells. Similarly, female BWF1 lupus mice have increased expression of interferon genes compared to age and sex- matched males. Estradiol regulates interferon-inducible genes differentially in healthy controls and in SLE, with higher induction of IFN-inducible genes in the SLE population. Plasma 17b-estradiol levels are significantly increased in female SLE patients compared to healthy females (p<0.01). Testosterone (100 pg/ml) significantly increased FoxP3 expression in CD4+CD25+hi cells from women with SLE (p< 0.03). Estradiol increases proinflammatory cytokines and positively correlated with plasma levels of IL-6/ IL-21 and miR21, miR25 and miR186.