bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transduction of retinoic acid-inducible gene 1 by Ebola virus-like particles enhances antigen-presentation
Authors: Warneke Paul , Braaten Jordan , Ngu Mee Kee Ester , Potter Hannah , Nelson Grace , Pottebaum Ethan , Martinez Osvaldo 
Published: January 2023

Abstract
Ebola virus (EBOV), a filovirus family member, is a highly pathogenic virus that causes Ebola hemorrhagic fever resulting in documented mortality rates in humans as high as 50%. EBOV virus-like particles (VLPs) are morphologically and biochemically similar to parental virus, yet they lack a genome and cannot replicate. We hypothesize that addition of a constitutionally active retinoic acid-inducible gene 1 (RIG-I) would enhance the ability of the EBOV VLPs to induce antigen-presentation from infected antigen-presenting cells. Expression of EBOV VP40 in 293T cells induces the spontaneous production of VLPs into the media supernatant and if expressed with EBOV glycoprotein (GP), will produce VLPs studded with the attachment GP. Recombinant chimeric constitutively active (ca)RIG-I-VP40 matrix and a nonfunctional mutant L58A (mu)RIG-I-VP40 matrix genes were constructed to produce VLPs containing constitutively active and nonfunctional RIG-I, respectively. Supernatant from 293Ts transfected with caRIG-I-VP40, muRIG-I-VP40 or VP40 along with GP expression plasmids were tested for the presence of VLPs. Western blotting of purified VLPs confirmed the presence of RIG-I in caRIG-I-VP40 and muRIG-I-VP40 VLPs. Monocyte-like THP-1 reporter cells were treated with nothing, VP40+GP, caRIG-I-VP40+GP and muRIG-I-VP40+GP VLPs as well as LPS and tested for induction of interferon signaling. CaRIG-I containing, but not muRIG-I containing VLPs induced interferon signaling as well as greater MHC I upregulation. Furthermore, CaRIG-I containing, but not muRIG-I containing VLPs induced greater levels of IL-2 production from treated mixed-lymphocyte reactions. Future studies will test the vaccine efficacy of caRIG-I containing VLPs.
Induction of Zika specific CD8 T cell responses in the placenta after heat shock protein gp96-Ig vaccination
Authors: Strbo Natasa , Romero Laura , Fisher Eva , Garcia Denisse 
Published: January 2023

Abstract
Heat shock protein (HSP) gp96 is a biological adjuvant that activates antigen-presenting cells and simultaneously delivers antigen specific peptides to MHC I for CD8 T cell activation. Here, we have transformed human embryonic kidney cell line (HEK-293) into powerful miniature osmotic pumps that continually secrete engineered form of gp96, gp96-Ig, along with its chaperoned antigens (ZIKV envelope immunogenic peptides) to induce ZIKV specific CD8+ cytotoxic T cell immune responses.
B6 females were mated with B6 or BALB/c males and gestation day (GD) 0.5 was determined by the presence of vaginal plug. At GD 7.5 one million of irradiated 293-gp96-Ig secreting cells were injected subcutaneously (SC). Mice were followed up until the end of pregnancy (GD 19.5–21.5) or humanely euthanized 5 days after vaccination (GD 12.5). Frequency of CD8+ T cells was determent by flow cytometry, immunohistochemistry and antigen, ZIKV envelope specificity was confirmed by in vitro ZIKV peptide stimulation and by intracellular cytokine staining.
We found that vaccinated mice have the same number of live-born pups as control mice without pathological changes in placenta or in fetus. This is the first finding that confirms the presence of gp96-Ig-ZIKV-induced CD8 T cells around arteries and larger blood vessels in the maternal part of the placenta, decidua. Furthermore, secreted gp96 vaccine induces very high frequency of ZIKV specific CD8 T cells in the maternal decidua (15%) without compromising placental or fetal viability.
In summary, we have succeeded in generating vaccine that is expressing gp96-Ig and ZIKV envelope protein and also we confirmed that vaccination with secreted gp96-Ig-ZIKA is safe and induces CD8 T cell responses in maternal decidua.
In vivo NKTT320-induced activation of invariant Natural Killer T-cells (iNKTs) and potential applications for use in AIDS pathogenesis modulation
Authors: Bond Nell G. , Yu Shan , Rout Namita , Tran Dollnovan , Szeltner Dawn , Schaub Robert , Kaur Amitinder 
Published: January 2023

Abstract
iNKTs are unique, innate T cells with an invariant TCRα chain that recognize glycolipids presented by nonpolymorphic MHC class-I-like CD1d molecules. Activated iNKTs rapidly secrete a range of cytokines and thus have diverse functions including potentiation of anti-tumor and pathogen-specific humoral and cellular immunity and resolution of inflammation. iNKT depletion and dysfunction are common in pathogenic HIV/SIV infection and may hasten AIDS progression. In this study, we tested a novel monoclonal antibody, NKTT320, that specifically binds to the iNKT cell TCRα as a tool for direct in vivo iNKT activation in Mauritian cynomolgus macaques (MCM).
Dose-escalation NKTT320 pharmacokinetic studies were performed in 11 SIV-negative MCM. Plasma NKTT320 levels were measured by ELISA while kinetics of iNKT activation and effect on other immune cell subsets were evaluated by flow cytometry.
NKTT320 plasma levels peaked between 6.6–47.2 μg/ml within 24 hours of a single IV doses of 100–1000 μg/kg and declined below detection by 4–6 weeks. NKTT320 administration resulted in rapid iNKT activation and proliferation of CD4+ more than CD8+ iNKTs, without non-specific T cell activation. Transient elevation of several plasma analytes (IL-2, IL-6, IL-17, MCP-1, MDC and IP-10) and increased proliferation of non-T immune cell subsets were observed suggesting downstream potentiation of innate and adaptive immunity. Repeated doses of NKTT320 increased iNKT responsiveness indicating that treatment did not lead to iNKT anergy.
NKTT320-mediated iNKT activation may be a useful therapeutic tool to preserve and enhance iNKT function, thereby preventing immunodeficiency and aberrant immune activation in pathogenic SIV infection.
Antigen targeting of Fc-receptors induces strong T cell responses in vivo independent of ITAM signaling but dependent on dendritic cell subsets
Authors: Lehmann Christian H. K. , Baranska Anna , Heidkamp Gordon F. , Heger Lukas , Neubert Kirsten , Lühr Jennifer J. , Hoffmann Alana , Reimer Katharina C. , Brückner Christin , Beck Simone , Seeling Michaela , Kießling Melissa , Soulat Didier , Krug Anne B. , Ravetch Jeffrey V. , Leusen Jeanette H.W. , Nimmerjahn Falk , Dudziak Diana 
Published: January 2023

Abstract
Dendritic cells (DCs) are important antigen presenting cells (APCs) and responsible for the induction of immune responses and preserving peripheral tolerance. We showed that targeting of antigens via C-type lectin receptors to different specialized DC subpopulations induced either CD4+ or CD8+ T cell responses in vivo. Fc receptors are also highly active in endocytosis enabling APCs to take up antigens in form of immune complexes. As they are expressed on various APCs, we aimed to identify responsible APCs for primary and secondary immune responses. Therefore, we assessed their expression in various organs and delivered antigens by specific recombinant antibodies. The targeting of the Fc receptors induced CD4+ and CD8+ T cell responses in a transgenic as well as a naïve system. Moreover, especially antigen delivery to the activating FcγRIV was superior in inducing CD4+ and CD8+ T cell responses at the same time, which could not be observed by classical targeting of the C-type lectin receptors DEC205 or DCIR2. Additionally, targeting of antigens to FcγRIV induced a pronounced CD4 helper response in naïve mice, whereas targeting to DCIR2 was inefficient. As FcγRIV is expressed on both major splenic DC subsets, we used this receptor to verify the subset intrinsic preference of DCs for CD4+ or CD8+ T cell responses. This was clearly shown by the induction of CD4+ T cell responses by splenic CD8− DCs, whereas splenic CD8+ DCs induced a CD8+ T cell response.
The induced naïve CD8+ T cell responses were functional relevant, as we could demonstrate efficient dose-dependent killing of target cells in vivo. Therefore, we suggest this strategy as useful tool for the induction of de novo as well as the modulation of immune responses for therapeutic applications.
Generation of highly functional cross presenting monocyte-derived dendritic cells with a new automated and closed electroporation system
Authors: Andreoni Cristina Conforti , Altmann Tobias , Bergschneider Eva , Heckötter Jessica , Birth Kai-Michael , Dzionek Andrzej 
Published: January 2023

Abstract
The value and safety of dendritic cell (DC) vaccines in cancer immunotherapy have been proven in several clinical studies using monocyte-derived dendritic cells (Mo-DCs). However, the parallel development of several different maturation and antigen-loading protocols might have led to cellular products with different therapeutic efficacy. Moreover a low immunological capability of the patients might cause the generation of functionally impaired DCs.
Functionality of Mo-DCs can be improved by mRNA electroporation, an effective method for introducing either antigens for MHC class I-mediated presentation (cross presentation) or other genes that might modulate the functional properties of Mo-DCs in a desired way. Yet, a standardized and reproducible GMP-compliant manufacturing of such cellular product requires instrumentation with specific characteristics not always provided by conventional technologies.
In order to address this need, we have developed a platform for automated manufacture of cellular products (CliniMACS Prodigy and CliniMACS Electroporator), enabling a reproducible and scalable generation of Mo-DCs and automated electroporation in a closed system that facilitates compliance with GMP requirements.
To validate the functionality of our system we first optimized transfection rate using GFP-mRNA. Then, using mRNA encoding immune relevant antigens we generated Mo-DCs able to stimulate autologous, antigen specific, cytotoxic T cells. Moreover, we successfully induce expression of genes that are responsible for main DC functions.
In summary, we developed an automated, large-scale, closed process that enables efficient transfection of Mo-DCs for antigen delivery and functional tuning
T Cell Antigen Discovery using Signaling and Antigen-presenting Bifunctional Receptors (SABRs)
Authors: Joglekar Alok , Leonard Michael Troy , Jeppson John , Bethune Michael T. , Baltimore David 
Published: January 2023

Abstract
Checkpoint inhibitors, cancer vaccines, and adoptive cell therapy exploit T cell mediated immune responses to cancers. Discovering the exact antigens targeted by T cell responses is important for their efficacy. Antigen discovery for ‘orphan’ T cells or TCRs has been a challenging prospect due to high number of possible pMHC specificities. Several current approaches to decipher antigen specificities require prior knowledge of antigen sequences, are unable to scale up, or require production of soluble TCRs. To overcome these drawbacks, we have developed chimeric receptors called Signaling and Antigen-presenting Bifunctional Receptors (SABRs) that allow identification of antigen-presenting cells. SABRs present display pMHC on their extracellular domain, which is recognized by an orphan TCR. Upon recognition, SABRs initiate signaling in the presenting cell using a CD3zeta signaling domain. We transduced reporter cells with SABRs presenting HLA-A2-restricted epitopes from MelanA and NY-ESO-1, and co-incubated them with target cells expressing their cognate TCRs, which resulted in signal transduction only upon correct pMHC-TCR pairing, allowing the presenting cells to express GFP. Second, we showed that SABRs displaying independently expressed peptide and MHC could function similarly. These receptors could present pulsed peptides or endogenously expressed proteins, allowing the uncoupling of peptide and MHC, while retaining their signaling capability. We are currently testing the use of SABR-based antigen libraries to identify novel antigenic specificities targeted by T cells in cancers, infectious diseases, and autoimmune diseases.
Determinants of T follicular helper cell development upon viral infections
Authors: Kuka Mirela , De Giovanni Marco , Maganuco Carmela Gianmaria , Cutillo Valeria , Cristofani Claudia , Di Lucia Pietro , Bono Elisa , Giustini Leonardo , Iannacone Matteo 
Published: January 2023

Abstract
Immune protection against viral infections relies on the generation of efficient B cell responses and neutralizing antibody (nAb) production. In order to reach full activation, B cells undergo several activation checkpoints in lymph nodes (LN) draining infections sites. In particular, crosstalk between antigen-specific B cells and T follicular helper (Tfh) cells is key to the formation of germinal centers and to the production of high affinity nAbs. However, some viruses (such as lymphocytic choriomeningitis virus [LCMV] in mice, and hepatitis B and C in humans) fail to induce early, potent nAb responses, and can establish persistent infections. We asked whether the lack of nAbs upon LCMV infection reflects a defect in T cell help to B cells. To address this question, we sought to analyze the generation and activation of Tfh cells in the context of LCMV infection as compared to VSV infection, a benchmark for effective nAb responses. Preliminary data show a striking compartmentalization of CD4 T helper responses. In particular, the vast majority of VSV-specific CD4+ T cells differentiated into Tfh and migrated to B cell follicles as early as three days after infection. By contrast, LCMV infection resulted in almost exclusive Th1 differentiation, with little or no Tfh induction. Interestingly, this was independent of the affinity of the TCR for the antigen, since the same LCMV glycoprotein antigen induced Tfh cell differentiation when expressed on a VSV backbone. We are currently utilizing intravital microscopy and gene-expression profile analyses to pinpoint the mechanisms underlying this reduced Tfh differentiation in the context of LCMV infection. Elucidating these mechanisms could pave the way to more efficient vaccination strategies.
HIV blocks Type I Interferon signaling through disruption of STAT1 phosphorylation.
Authors: Tran James Thien , Nguyen Nam V. , Sanchez David Jesse 
Published: January 2023

Abstract
Although a considerable amount of research has been undertaken to find new strategies to treat acquired immunodeficiency syndrome (AIDS), which is caused by human immunodeficiency virus (HIV), it still remains a global health complication. Type 1 Interferon (IFN) is well-known to activate the antiviral state via the JAK/STAT pathway. However, HIV is able to bypasses multiple antiviral responses due to encoded proteins that disrupt various steps in the stimulation of the IFN-induced antiviral state. We hypothesize that HIV proteins block the antiviral state by disrupting the JAK/STAT signaling pathway even in the presence of exogenous IFN-α. We found that HIV proteins inhibit the phosphorylation of STAT1 in CD4+T cells. Furthermore, we found a reduction of phosphorylated STAT1 after IFN-α stimulation in cells expressing Vpu and Nef. On the other hand, the expression levels of Tyk2 and total STAT1 are not affected by Vpu or Nef. We also observed a reduction in the antiviral effect induced by IFN-α using infection of VSV-GFP in a Vpu stable cell line. In conclusion, HIV IFN-α signaling via expression of Vpu and Nef allowing for reduction in antiviral state establishment by IFN-α. In the future, we plan to assess Vpu and Nef inhibition of the IFN-α mediated antiviral state in macrophages to see if it is similar to the effect in T cells.
Gammaherpesvirus infected B cells display abnormal repertoire
Authors: Zelazowska Monika Anna , Dong Qiwen , Plummer Joshua , Mu Yunxiang , MacCarthy Thomas , Krug Laurie T. , McBride Kevin M. 
Published: January 2023

Abstract
Gammaherpesviruses (γHV) are oncogenic viruses that establish lifelong latency in B cells. Chronic infection with human γHV, Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) is associated with the development of lymphoproliferative diseases, lymphomas and other types of cancer. The murine equivalent of human γHV, MHV68, is widely used as an animal model for γHV infection.
Primary infection is associated with mucosal tissue replication, expansion in germinal center (GC) B cells and chronic persistence in host’s memory B cells. The GC compartment undergoes somatic hypermutation (SHM), class switch recombination (CSR) and repertoire selection which generate long lived antibody-secreting plasma cells and memory B cells. It is not clear what impact γHV infection has on B cell differentiation, affinity maturation and clonal proliferation.
We isolated and comparatively analyzed the B cell repertoire of infected and non-infected GC cells from individual mice. This was done by sequencing repertoire from both the single cells and GC populations using NGS high-throughput sequencing. We determined the effect of infection on diversity, selection, mutagenesis and expansion of clones within the GC population of a mouse. Our data indicate that repertoire from MHV68 infected B cell are distinct from non-infected GC cells and is characterized by unique clones, repertoire bias and inappropriate expansion of certain clones. Moreover, both B cell populations share CDR3 variants only on minimum level. Thus, our findings demonstrate that viral infection cause a subversion of GC mutagenic and selective processes which result in a skewed repertoire.
Ikaros establishes an epigenetic program required for proper two-signal activation of naive B cells
Authors: Rodriguez Princess D. , Schjerven Hilde , Amiel Eyal , Frietze Seth 
Published: January 2023

Abstract
While there are many different types of autoimmune diseases, a common feature of chronic inflammatory diseases is the production of autoantibodies by B-cells. We and others have shown in mouse models that loss-of-function mutations in the transcription factor Ikaros (Ik) result in a hyper-reactive (HR) B-cell phenotype. In particular, naïve B-cells containing a targeted deletion in the Ikzf1 zinc finger domain (Ikzf1ΔF4/ΔF4) produce large quantities of autoantibodies and proliferate in the absence of a costimulatory signal. Ik is required for early lymphoid cell development and has been shown to regulate gene expression directly or through the recruitment of chromatin remodeling complexes. However, the epigenetic and transcriptional regulation by Ik in mature B-cells and its role in B-cell HR remain unknown. We hypothesized that Ik functions to establish and maintain an epigenetic program required for proper two-signal activation in naïve B-cells. We have generated whole transcriptome profiles of wildtype and Ikzf1ΔF4/ΔF4 cells and defined the gene expression program required for B-cell activation. A comparison of these profiles revealed distinct deficiencies in genes associated with the HR phenotype. Specifically, a variety of cell signaling pathways are derepressed in Ik-mutant B-cells. In addition, we have performed Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), over the course of B-cell activation, to explore the altered chromatin structure in Ik-mutant B-cells. Together these results revealed transcriptional pathways required for proper B-cell activation and demonstrate how mutations in Ik contribute to B-cell HR.
Genes associated with ARID3a expression in B Lymphocytes from Systemic Lupus Erythematosus patients
Authors: Garton Joshua W. , Pelikan Richard , Ratliff Michelle , James Judith A. , Gaffney Patrick , Webb Carol F. 
Published: January 2023

Abstract
Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by overproduction of autoantibodies by B cells and the loss of tolerance to nucleic acids. Our previous studies demonstrated that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein associated with immunoglobulin transcription, is more abundant in SLE B lymphocytes than in healthy control B cells. ARID3a expression in SLE patient blood occurred in naïve B cell subsets which do not express ARID3a in healthy controls. Importantly, increased numbers of ARID3a+ B cells in SLE correlated with increased disease activity in patients. Our recent data indicated that ARID3a is critical for the expression of the key inflammatory cytokine, interferon-alpha (IFNa), in B cells and other blood cells. ARID3a+ B cells appear to denote a new kind of B effector cell that can induce IFNa expression in other cell types. We hypothesize that ARID3a is an important contributor to increased disease activity in SLE patients. Therefore, it is important to better define the characteristics of these cells. ARID3a is bimodally expressed, such that only a fraction of the cells within any B cell subset express ARID3a at a given time. Our current studies took advantage of this bimodal expression using single-cell technology to perform RNA-seq analyses of ARID3a+ and ARID3a− SLE B cells to identify differentially expressed genes associated with ARID3a. To date, ARID3a expression is correlated with innate immune response genes and epigenetic regulators. We predict these studies will be informative regarding how ARID3a expression contributes to disease pathogenesis in patients with SLE, and will provide novel information defining a new subset of effector B cells.
Follicular CD8 T cells function in germinal center reactions to promote autoantibody-mediated disease.
Authors: Valentine Kristen M. , Mullins Genevieve , Pinney James M. , Hoyer Katrina K. 
Published: January 2023

Abstract
CD8 T cell function, unlike CD4 T cells, in antibody-mediated autoimmune disease remains largely unexplored. CD8 T cells act as either cytotoxic or helper-like cells during autoimmunity. CD4 T follicular cells (Tfh) interact with B cells to promote proliferation, activation and antibody class switching. CXCR5+PD-1hi CD8 T follicular (Tfc) cells localized within the germinal center resemble CD4 Tfh cells in multiple models of spontaneous autoantibody-mediated disease, including IL-2 deficient (IL-2-KO), Scurfy and MRL/MpJ-FASlprmice. CD4 and CD8 T cells activated with NP-KLH synergistically induce B cell class switching to IgG1 IgG2a, IgG2b, and IgG3 in vivo. RNA-sequencing of IL-2-KO CD8 Tfc and IL-2-KO CD4 Tfh cells identified 478 differentially expressed genes unique to CD8 Tfc cells. These data revealed an increased expression of genes associated with CD8 Tfc cell function including il21, ifnγ, prf1. IL-21 produced by CD4 Tfh cells but not CD8 Tfc cells promotes total IgG class switching demonstrated by in vitro cultures of activated autoimmune CD8 Tfc or CD4 Tfh cell supernatant incubated with IL-21R-KO B cells. IFNγ x IL-2-KO mice develop both CD4 Tfh and CD8 Tfc cells correlating with autoantibody production. In vitro culture assays demonstrate that IFNγ produced by CD8 Tfc cells promotes B cell class switching to. Our results indicate that CD8 Tfc cells contribute to autoimmune disease synergistically with CD4 Tfh cells to induce B cell differentiation and antibody class-switch. We are continuing to explore the unique functions of CD8 Tfc cells on germinal center interactions.