bluuebunny/crossref_metadata_2025 huggingface dataset:
Utilization of High-Frequency Irreversible Electroporation (H-FIRE) to modulate the tumor microenvironment and promote systemic immune system activation in breast cancer
Authors: Allen Irving C. , Ringel-Scaia Veronica , Coutermarsh-Ott Sheryl , Brock Rebecca , Lorenzo Melvin , White Natalie , Oestreich Kenneth J. , Verbridge Scott , Davalos Rafael
Published: January 2023

Breast cancer is the most common malignancy among women, where almost 1.7 million new patients worldwide are diagnosed annually. Despite promising treatments for breast cancer, metastatic disease and recurrence remain significant challenges. There is no highly effective cure currently available once metastatic disease has developed. Thus, new therapeutic paradigms and treatment strategies are direly needed. High Frequency Irreversible Electroporation (H-FIRE) is a particularly novel and emerging therapeutic approach for tumor ablation. This technique uses a series of high-frequency, bipolar electric pulses applied through electrodes inserted directly into the targeted tumor to induce cancer cell death. Our overarching hypothesis predicts that local treatment of the breast tumor with H-FIRE will significantly stimulate the innate and adaptive immune system, resulting in improved survival outcomes. To test this hypothesis, we utilized a mouse 4T1 mammary tumor model. Here, we show that H-FIRE treatment of the primary tumor results in near complete ablation and a shift in the local tumor microenvironment from immunosuppressive to pro-inflammatory. This shift results in the recruitment of increased numbers of antigen presenting cells and T lymphocytes. Local H-FIRE treatment also significantly reduces 4T1 metastases in animals with an intact immune system, indicating increased engagement of a systemic anti-tumor immune response and improved activation of the adaptive immune system. We anticipate that this novel tumor ablation technology will improve conventional treatment strategies and complement emerging immunomodulatory approaches targeting primary tumors, metastatic lesions, and preventing recurrence.
A novel and easy-to-use approach for assessment of chimeric antigen receptor (CAR) modified immune cell cytotoxicity
Authors: Xiong Wei , Weber Nikki , Liu Dongfang
Published: January 2023

Cancer immunotherapies have shown promising results for treating various cancers. One such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), thus combining tumor antigen specificity with immune cell activation in a single receptor. Currently, assessment of this CAR-modified immune cell cytotoxicity is limited by conventional tools (e.g., standard Chromium-51 release assay), which are time-consuming, costly, and labor-intensive. In this study, we developed a novel, easy-to-use, and non-radioactive approach for assessment of CAR-modified immune cell cytotoxicity on the basis of luciferase bioluminescent signal. After a 4- hour incubation of CAR-modified immune cells in a 96-Well optical-bottom microplate, which was pre-seeded with targeted cell stablely expressed eGFP -firefly luciferase fusion gene (GFP-FFluc), the chemical bioluminescent signal of GFP-FFluc was quantified by a fluorescent microplate reader. The positive correlation between FFLuc signal and number of target cells was observed. The dose-dependent specific lysis is comparable with Chromium-51 release assays. The cytotoxicity activity measured by this approach can be further quantified under a common inverted fluorescence microscope to evaluate the morphology and dynamics of CAR-modified immune cells. In conclusion, this newly developed approach provides the scientific community with a powerful tool to study the CAR-modified immune cell cytotoxicity.
Targeting HLA-E for prostate cancer immunotherapy
Authors: Frueh Klaus , Verweij Marieke , Hansen Scott , Mansouri Mandana , Nair Sujit , Malouli Daniel , Tewari Ashutosh K. , Uebelhoer Luke , Ventura Abigail , Selseth Andrea , Axthelm Michael , Bhardwaj Nina , Picker Louis J.
Published: January 2023

Genetically modified strain 68-1 rhesus cytomegalovirus (RhCMV)-derived vaccine vectors uniquely elicit CD8+ T cells recognizing peptides presented by non-polymorphic MHC-E instead of conventional MHC-I molecules. Since MHC-E (HLA-E in humans) is a ligand for inhibitory receptors on NK cells, the upregulation of MHC-E is a known immune evasion strategy for both cancers and intracellular pathogens. Indeed, using tissue arrays we observed that early stage prostate cancer tissues express high levels of HLA-E, as shown by immunohistochemistry, whereas healthy prostate samples were negative. To determine whether MHC-E-restricted T cells recognizing prostate antigens could be used to target prostate cancer we inserted rhesus prostatic acid phosphatase (rhPAP) into RhCMV. Upon inoculation of rhesus macaques (RM) we observed a T cell response comparable to heterologous antigens suggesting that CMV-based vectors excel at eliciting T cell responses to tumor antigens, including self-antigens. Furthermore, all CD8+ T cells recognized rhPAP either in the context of MHC-II or MHC-E, but not MHC-I. Importantly, MHC-E-restricted CD8+ T cells recognized PAP and MHC-E-expressing K562 cells suggesting that endogenous PAP-derived peptides can be loaded onto MHC-E. We further demonstrate that PAP and HLA-E-positive human prostate adenocarcinoma cells stimulated PAP-specific rhesus CD8+ T cells restricted by MHC-E. We conclude that prostate cancer cells load highly conserved HLA-E molecules with prostate antigen-derived peptides that can be targeted by HLA-E-restricted CD8+ T cells thus exploiting a previously unexplored immunological vulnerability.
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

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

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

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

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

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

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

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

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

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

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

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

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.
Activation of the Stimulator of Interferon Gene Protein is Involved in the Etiopathogenesis of Sjogren’s Syndrome
Authors: Papinska Joanna Aleksandra , Bagavant Harini , Gmyrek Grzegorz B. , Sroka Magdalena , Tummala Saigiridhar , Fitzgerald Kate A. , Deshmukh Umesh S.
Published: January 2023

Activation of innate immune responses and excessive production of type I Interferons (IFNs) plays a critical role in the pathogenesis of Sjögren’s syndrome (SS), a chronic autoimmune disorder affecting the exocrine salivary glands (SG). Recognition of nucleic acids by cytosolic nucleic acid sensors is a major trigger for type I IFN production. Several cytosolic DNA sensors interact downstream with the stimulator of interferon gene (STING) protein and induce type I IFNs. Although STING has been implicated in several interferonopathies, its role in SS is not known.

To investigate whether STING activation is involved in the etiopathogenesis of SS, in this study, female C57BL/6 mice were injected with a STING agonist, dimethylxanthenone-4-acetic acid (DMXAA) and mice were followed for the development of SS. Systemic DMXAA treatment rapidly induced the expression of Ifnb1, Il6, and Tnfa in the SG, and these cytokines were also elevated in circulation. In contrast, increased Ifng gene expression was only detected in the SGs. The type I innate lymphoid cells present within the SG were the major source of IFNγ, and their numbers increased significantly within 3 days of DMXAA treatment. STING expression in SG was mainly observed in the ductal and interstitial cells. In primary SG cells, DMXAA activated STING, which caused phosphorylation of TBK1 and IRF3, and induced IFN-β production. The DMXAA-treated mice developed autoantibodies, sialoadenitis, and SG hypofunction.

Our study demonstrates that activation of STING pathway holds the potential to initiate SS. Thus, apart from viral infections, conditions that cause cellular perturbations and accumulation of host DNA within the cytosol should also be considered as possible triggers for SS.
Effect of combustible tobacco product preparations on neutrophil-like cells
Authors: Arimilli Subhashini , Cutno D’Quan Ke’shawn Lamar , Prasad G.L.
Published: January 2023

Chronic exposure to cigarette smoking causes inflammation and immune suppression, which are known to contribute to smoking-related diseases and increased susceptibility of smokers to microbial infections. Neutrophils play an essential role in innate immune responses to protect against microbial infections. However, there is limited information on how smoking impacts innate immunity, particularly neutrophil functions. The use of neutrophils in research is limited due to their short lived nature. Therefore, alternate sources for neutrophil-like cells have been sought. Treatment with DMSO, results in differentiation of the HL60 cells to neutrophil-like cells, which provides an alternate model for assessment of certain neutrophil functions. In this study, we have differentiated HL60 cells with DMSO to neutrophil-like cell phenotype. The differentiated cells were characterized by the expression of CD11b and phagocytosis, and assessed the effects of exposure to different tobacco products. We assessed the effect of combustible cigarette whole smoke conditioned medium (WS-CM) and smokeless tobacco (ST) extracts, prepared from reference tobacco products, on the neutrophil-like cells on cell migration and phagocytic function. A dose-dependent decrease in the migration and phagocytosis was observed on treatment with WS-CM, but not with ST extracts. These findings suggest that neutrophil-like cells can be utilized to assess the effects of combustible and non-combustible tobacco products. Further, these results show that combustible and non-combustible tobacco products differentially impact neutrophil functions in this model.
Lysophosphatidic acid strongly protects acute liver injury caused by acetaminophen in an experimental animal model
Authors: Bae Geon Ho , Lee Sung Kyun , Kim Hyung Sik , Lee Mingyu , Lee Ha Young , Bae Yoe-Sik
Published: January 2023

The effect of lysophosphatidic acid (LPA) against experimental acetaminophen (APAP)-induced acute liver injury was investigated. LPA administration elicited strongly protective effects against APAP-challenged acute liver injury, showing attenuated liver damage, liver cell death, and AST and ALT levels. APAP overdose-induced mortality was also significantly attenuated by LPA administration. On the mechanism involved in LPA-induced protection against acute liver injury, LPA not only markedly increased the glutathione level but also strongly blocked the APAP challenge-elicited phosphorylation of JNK, ERK, and GSK3β, which are involved in the pathogenesis of acute liver injury. Moreover, LPA decreased the production of TNF-α and IL-1β in an experimental liver injury animal model. Mouse primary hepatocytes express LPA1,3–6, and injection of the LPA receptor antagonist KI16425 (an LPA1,3-selective inhibitor) or H2L 5765834 (an LPA1,3,5-selective inhibitor) did not reverse the LPA-induced protective effects against acute liver injury. In conclusion, our findings indicate that LPA can block the pathogenesis of APAP-induced acute liver injury by increasing the glutathione level but decreasing inflammatory cytokines in an LPA1,3,5-independent manner. Therefore, we suggest that LPA can be considered as a possible therapeutic agent for drug-induced liver injury.
Suppression of CD8+ T cell and Neutrophil Function Distinguishes Ruxolitinib Treatment from IFNγ Neutralization in Hemophagocytic Lymphohistiocytosis
Authors: Albeituni Sabrin H. , Verbist Katherine , Tedrick Paige , Tillman Heather , Nichols Kim
Published: January 2023

HLH is an often-fatal cytokine storm syndrome characterized by an ineffective yet overactive immune response. CD8+ T cells and IFNγ are thought to be the primary effectors driving disease; however, outcomes for HLH patients remain suboptimal despite therapies that target T cells or IFNγ. As many of the cytokines amplified in HLH signal through the JAK-STAT pathway, we examined the therapeutic potential of the JAK1/2 inhibitor ruxolitinib in HLH. For mechanistic insight, we also compared its effects to IFNγ neutralization (αIFNγ). Using complimentary murine models (LCMV-infected perforin-deficient mice to model “primary” or inherited HLH and administration of CpG+αIL10R to model “secondary” non-inherited HLH), we found that HLH-associated cytopenias were ameliorated by either agent, but cellular inflammation was improved only by ruxolitinib. Consistently, ruxolitinib was also superior to αIFNγ in reducing the number and size of hepatic inflammatory foci and decreasing the numbers and cytokine-producing capacity of tissue-infiltrating CD8+ T cells and neutrophils. Surprisingly, ruxolitinib-induced reductions in pro-inflammatory cytokine levels and enhancements in survival were sustained despite discontinuation of the drug. However, such was not the case following discontinuation of αIFNγ. Fatality in these latter animals was reversed upon the simultaneous depletion of neutrophils. Together, these data reveal that the hematological parameters of HLH are IFNγ-dependent, while tissue immunopathology results from the activity of numerous cytokines that drive expansion and activation of CD8+ T cells and neutrophils. These data further support the incorporation of ruxolitinib into future HLH clinical trials.
Exogenous Lipocalin2 facilitates the upregulation of thermogenic and beige/browning markers in murine white adipocytes
Authors: Meyers Keya R. , Lopez Maria , samuels janaiya , Rayalam Srujana , Shashidharamurthy Rangaiah
Published: January 2023

Lipocalin2 (Lcn2) belongs to a small molecular weight lipocalin subfamily of secretory proteins that binds to small hydrophobic molecules. Recent studies have shown that Lcn2 is highly upregulated by adipocytes and is categorized as a new adipokine with a role in innate immunity and metabolic disorders, including obesity. However, the exact role Lcn2 plays in the mediation and progression of obesity has yet to be elucidated. Herein, we have investigated the role of exogenous Lcn2 in inducing the browning of white adipocytes in vitro using the 3T3-L1 murine adipocyte cell line. Treatment with exogenous recombinant Lcn2 (rec-Lcn2) resulted in the upregulation of thermogenic and beige/brown markers (UCP1, PGC-1α, PPARγ, PRDM16, ZIC-1 and TBX1) in mature adipocytes. Additionally, rec-Lcn2 increased mitochondrial activity in 3T3-L1 cells. Further, rec-Lcn2 decreased the pre-adipocyte differentiation resulting in the attenuation of adipogenesis as evidenced by reduced lipid content accumulation. These results suggest that Lcn2 is a naturally occurring adipokine, and may serve as an anti-obesity agent by both decreasing adipogenesis in white adipocytes and inducing the transdifferentiation of white to brown-like beige adipocytes. Therefore, Lcn2 could be a potential target to attenuate/prevent obesity.
Glucocorticoids inhibit group 3 innate lymphocyte IL-22 production
Authors: Zenewicz Lauren A. , Seshadri Sudarshan , Pope Rosemary L.
Published: January 2023

Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). IBD is the manifestation of a dysregulated mucosal immune response, usually against commensal bacteria, involving many different immune cells. GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone down-modulates both innate and adaptive immune cell activation. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides and mucins. The effects of dexamethasone on T cell function are well described, however, it is not known if dexamethasone modulates the critical function of ILC3s or if dexamethasone regulates the cytokine IL-22. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23-mediated IL-22 production in homeostatic and activated human and mouse ILC3s. Dexamethasone did not modulate the IL-23 canonical signaling pathway of JAK2/STAT3, but did suppress phosphorylation of IkBa, an important regulator in NF-kB activation. These findings implicate NF-kB as a regulator of IL-22 production in ILC3s and have likely repercussions on GC treatment of IBD patients.
Selective electrical stimulation of vagus nerve induces specific cytokine response.
Authors: Datta-Chaudhuri Timir , Tsaava Tea , Addorissio Meghan , Bouton Chad , Tracey Kevin J. , Chavan Sangeeta S.
Published: January 2023

Neuro-immune communication and neural circuits regulating immunity have been therapeutically explored in preclinical models and successfully in recent human clinical trials. Implantable bioelectronic devices that stimulate the inflammatory reflex are effective in suppressing cytokine production during endoxtoxemia and other models of infection and injury. Whether vagus nerve stimulation can enhance cytokines was previously unknown. Here we analyzed serum cytokine levels in response to electrical stimulation of the vagus nerve using different pulse parameters. The cervical vagus nerve was isolated in adult male Balb/C mice and subjected to asymmetric charge-balanced stimulation using bipolar cuff electrodes. Animals were stimulated for 4 min using the following parameters: frequency 30–200 Hz, amplitude 50–750 μA, pulse width 50–250 μs. After 2 hrs, blood was collected and serum levels of TNFα, IL-6, IL-10, IL-12, and KC/GRO determined. Serum cytokine levels changed significantly (p<0.0001–0.05) and exhibited tuning curve properties when subjected to varying electrical pulse parameters, suggesting selective recruitment of efferent or afferent fibers that regulate specific cytokine production. Further insight into the nature of the neural circuits was ascertained through unilateral and bilateral vagotomies, distal and proximal to the stimulation location. Together, these studies demonstrate that systemic cytokine levels can be modulated by selectively stimulating the vagus nerve using specific combinations of frequency, amplitude, and pulse width. This study was funded in part by DARPA (HR0011-15-2-0016) and NIH (1R35GM118182-01).
IL-2 enhances IFNγ signals in subpopulations of T and B lymphocytes from treatment naive relapsing remitting multiple sclerosis (RRMS) patients.
Authors: Pope Brandon J. , Sharma Vishal , Boland Molly , Meador William , Bridges S. Louis , Raman Chander
Published: January 2023

Multiple sclerosis (MS) is the most common demyelinating disorder where most patients exhibit an undulating relapsing remitting course of disease progression (RRMS). Dysregulated effector and regulatory arms of the adaptive immune system contribute to disease progression and severity. Low dose interleukin 2 (IL-2) therapy is being considered for the treatment of autoimmunity to control adaptive effector responses and promote regulatory T cell (Treg) activity. In RRMS, others have shown that IFNγ responsiveness leads to loss of Treg function. We tested the possibility that IL-2 signals will dampen IFNγ dependent STAT activation in subpopulations of effector and regulatory T and B lymphocytes in a disease specific context. We stimulated PBMC from treatment naive RRMS or as a disease control, RA patients, with IFNγ and/or IL-2. Using a multi-parameter phospho-flow approach we evaluated the activation of STAT1, STAT3, and STAT5 in each subpopulation of T and B cells. The data was analyzed using t-SNE and SPADE visualization tools to interrogate changes in the activation and proportion of cell populations. We found IFNγ stimulation broadly induced greater activation of STAT1 in MS than in RA within multiple lymphocyte populations. IL-2 co-stimulation further increased IFN-γ activation in MS but not in RA cell populations, including in Tregs. Our observations suggest that in MS (1) there are enhanced IFNγ induced effector responses and (2) IL-2 signals synergize with IFNγ induced STAT1 activation. Thus, low dose IL-2 as a therapy for MS is likely to further exacerbate RRMS pathogenesis.
Soluble TIR-8 neutralizes anti-inflammatory effects of IL-37 in Crohn disease patients
Authors: Ahmad Ali , Samarani Suzanne , Abulkhair Ayoub , Jantchou Prevost , Faure Christophe , Deslandres Colette , Amre Devendra
Published: January 2023

Toll-IL-1Receptor (TIR)-8 is an atypical member of the IL-1 receptor family (IL-1RF). Recently, it was shown to act as the second sub-unit of the receptor for IL-37, the first being IL-18Rα chain. IL-37 is a potent anti-inflammatory cytokine belonging to the IL-1 family that exerts anti-inflammatory, immune deviating and metabolic effects. We found that the cytokine concentrations are increased in the circulation of Crohn disease (CD) patients compared with healthy controls. However, the expression of TIR-8 was decreased on the surface of different peripheral blood mononuclear cells in the patients. Furthermore, the concentrations of soluble TIR-8 were also increased in the circulation of the patients. Our results show that metalloproteinase inhibitors prevent shedding of TIR-8 from the cell surface. Soluble TIR-8 inhibits anti-inflammatory effects of IL-37 in human cells. When soluble TIR-8 was added in different concentrations, in the presence and absence of IL-37, to LPS-stimulated THP-1 cells, it inhibited the anti-inflammatory effects of the cytokine on the secretion of TNF-α from these cells. The inhibition was exerted in a dose-dependent manner. Taken together, these results suggest that IL-37 is neutralized in CD patients by TIR-8 shedding, which makes immune cells less responsive to the cytokine. Furthermore, soluble TIR-8 also neutralizes anti-inflammatory effects of the cytokine and contributes towards a pro-inflammatory state in these patients. Measures aimed at restoring anti-inflammatory effects of IL-37 by inhibiting shedding of TIR-8 and/or neutralizing its soluble form may be helpful in attenuating inflammation in CD patients.
More than a decoy receptor? Interleukin 22 Binding Protein (IL-22BP) in bacterial pneumonia
Authors: Trevejo-Nunez Giraldina , Castillo Patricia , Chen Kong , Kolls Jay K.
Published: January 2023

IL-22 is secreted by lymphoid cells such as Th17 cells and ILC3s. The IL-22 receptor is composed of IL-22Ra1 and IL-10Rbeta. In the lung, the IL-22R localizes in the upper airway epithelial cells. We previously reported that IL-22 contains pneumococcal lung infection due to hepatic activation of C3 and improved cellular phagocytosis. IL-22 also binds to a natural antagonist, known as IL-22BP or IL-22Ra2. In vitro, IL-22BP binds to IL-22 with high affinity, competing with IL-22R and neutralizing IL-22 activity. Studies have shown that IL-22BP controls tumor development in the colon, regulates inflammation in autoimmune encephalitis and promotes bacterial uptake in Peyer’s patches. However, it is unclear whether these multifaceted effects associated with IL-22BP are due solely to IL-22 binding and neutralization, given discrepant phenotypes in IL-22−/− versus IL-22BP−/− mice. Furthermore, surprisingly little is known about IL-22BP in the lung. We therefore interrogated the role of IL-22BP in a mouse model of bacterial pneumonia. In naïve lungs, we saw that IL-22BP is mostly expressed in alveolar and airway epithelial cells. IL-22BP−/− mice were less susceptible than controls to Streptococcus pneumoniae infection. This resistance was not due to differences in cellular recruitment, neutrophil phagocytosis or chemokine expression. RNA-Seq analyses of total lung tissue that compared infected IL-22BP−/− cohorts versus wild type revealed differential expression of several genes in the mitochondrial oxidative phosphorylation pathway. These data were strikingly different from prior findings made in IL-22−/−mice, leading us to speculate that IL-22BP may regulate mitochondria metabolism and have functions beyond the IL-22 system.