bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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&lt;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 &lt;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&lt;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

Abstract
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 (&gt;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&lt;0.0001) and SLE group (Pearson r =0.66, p&lt;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&lt;0.0001) than that of those above 50 year (Pearson r =0.57, p&lt;0.0001). This observation was also consistent in SLE patients (Pearson r = 0.76, p&lt;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

Abstract
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

Abstract
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 &gt;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

Abstract
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, &gt;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.