bluuebunny/crossref_metadata_2025 huggingface dataset:
Organ-on-Chip Meets Immunology: Let’s Start with the Lungs
Authors: Benam Kambez Hajipouran
Published: January 2023

Organs-on-chips are biomimetic, microfluidic, cell culture devices created with microchip manufacturing methods that contain continuously perfused hollow microchannels inhabited by living tissue cells arranged to simulate organ-level physiology. By recapitulating the multicellular architectures, tissue-tissue interfaces, chemical gradients, mechanical cues, and vascular perfusion of the body, these devices produce levels of tissue and organ functionality not possible with conventional 2D or 3D culture systems. They also enable high-resolution, real-time imaging and in vitro analysis of biochemical, genetic and metabolic activities of living human cells in a functional human tissue and organ context. Here, we discuss development of ‘Human Lung Small Airway-on-a-Chip’ – an engineered cell-by-cell reconstituted microfluidic device with living human tissues to model airway inflammation in the context of debilitating lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). In addition, we will present our preliminary data on recreating a hematopoietically active bone marrow niche in vitro (‘BM-on-a-Chip’) for microphysiological integration with the Small Airway-on-a-Chip to emulate innate immune cell egress into circulation (from the BM) and their recruitment to the lung airway.
Expanding the capabilities of the Maxpar Direct Immune Profiling Assay with additional markers, and customization of the analysis model in Maxpar Pathsetter
Authors: Li Stephen K.H. , Mujib Shariq , Cohen Michael , Yao Huihui , Majonis Daniel , Loh Christina
Published: January 2023

Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies. CyTOF can resolve more than 50 parameters in a single panel without the need for compensation, making it an ideal solution for routine enumeration of immune cells. The Maxpar® Direct™ Immune Profiling System is a sample-to-answer solution for human immune profiling using mass cytometry. The Maxpar®Direct™ Immune Profiling Assay™ is an optimized 30-marker panel contained in a dry single-tube format for human whole blood or PBMC staining, and samples are acquired on the Helios™ system. Maxpar Pathsetter™ is an automated software that reports cell counts, percentage calculations, and staining intensity. It also produces graphical elements such as dot plots and a Cen-se′™ graph for 37 immune cell populations. The panel can be tailored by adding markers to open channels, the Maxpar Pathsetter model can then be customized to measure expression markers on existing classified populations or identify additional immune cell subsets.

We present data where the Maxpar Direct Immune Profiling Assay is used as a core immunophenotyping panel and additional markers are added to create a nearly 50 marker panel. Added markers are used to identify MDSCs, further classify existing cell populations, and measure immuno-oncology related markers including OX40, TIM-3, Fas, PD-1, PD-L1, ICOS, and TIGIT. We demonstrate how the Maxpar Pathsetter model is modified to incorporate the added markers.

The ability to customize the Maxpar Pathsetter model and expand the Maxpar Direct Immune Profiling Assay allows for flexibility of the system. It allows researchers to have a streamlined solution for broad immune profiling using mass cytometry.
Immune monitoring for immuno-oncology applications
Authors: Chattopadhyay Pratip K , Lomas Woodrow E , Gao Guo-Jian , Li Na , Saksena Suraj
Published: January 2023

The immunotherapy revolution has spurred the development of many new drugs and drug regimens for patient treatment. A key challenge is to identify the factors that drive patient toxicities and responses to treatment, with a particularly acute need for predictive biomarkers that can discriminate patients destined to respond and fail treatment. Technologies to interrogate immune cells are now readily available, but important gaps remain in their application, which limit the full realization of the promise of precision oncology.

High parameter flow cytometry is a gold-standard but is limited by difficulty of panel design and lack of standardization. We present Color Wheel, a panel design tool, which builds optimized antibody panels based on the user’s instrument. These optimized panels have been manufactured in a dried, ready to use format to drive workflow and assay standardization. Initial results demonstrate excellent concordance between the dried and liquid versions of the high parameter multicolor panel(s).

Molecular cytometry represents an exciting new approach to high dimensional immune analysis because it can measure at least 102 proteins and 400 mRNA targets simultaneously per cell. An important application gap for this technology is lack of data indicating the sequencing depth needed for adequate resolution. Here, we present results from a molecular cytometry experiment sequenced deeply, and then bioinformatically sub-sampled at different levels to identify the minimum level of sequencing needed for clear identification of cells, which can serve as a reference guide for users to save time and cost in their experiments. We also present preliminary data generated using dried version of molecular cytometry panel(s).
High throughput subpopulation signaling analysis: protocol considerations for multiplexed intracellular flow cytometry
Authors: MacDonald Robert J , Manning Christopher , Alonzo Emily
Published: January 2023

While much of our knowledge of signaling pathways has come from bulk analytical methods such as western blotting, more granular techniques enable us to deepen our understanding of how signaling profiles may vary in heterogeneous cell populations. Flow cytometry offers a high-throughput avenue for subpopulation signaling analysis. Combining antibodies against extracellular CD markers with those against intracellular signaling proteins or post-translational modifications enables researchers to explore variations in signaling profiles by cell type.

Performing a staining protocol that allows for accurate performance of antibodies against both extracellular and intracellular epitopes is key. Manufacturers commonly validate antibodies using specific protocols; straying from these may lead to drastically different experimental results and conclusions. Antibodies against CD markers, for example, are often validated for use on live cells, and may perform poorly on fixed and permeabilized cells. Compromised CD marker detection may lead to inaccurate gating and thus inappropriate subpopulation analyses. In turn, this can lead to anomalous conclusions about the signaling profiles of subpopulations of interest.

In order to mitigate the risk of such occurrences, we present a general guide to protocol considerations when performing multiplexed intracellular flow cytometry through the lens of immune cell stimulation. Highlighting subpopulations of primary mouse immune cells treated with 2′3′-cGAMP, we demonstrate that CD marker staining protocol variation can lead to erroneous subpopulation characterization, which can then impact interpretation of innate immune signaling profiles within these populations.
Development of a novel model: Using songbirds to elucidate the pro-neurogenic role of microglia in healthy and natural neurodegenerative states
Authors: Larson Tracy A , Wolkoff Matthew E , Tokareva Kate , Cole Marianne Marritt , Brenowitz Eliot A
Published: January 2023

Inflammatory cells like microglia exert beneficial effects on neuronal survival, synaptic maintenance, and circuit plasticity. Most research has focused on neural injury or insult; roles in normal development and homeostasis remain less clear. To characterize the behavior of microglia under non-pathological conditions, we exploit seasonal death and replacement of neurons in a sensorimotor brain region responsible for singing behavior in white-crowned sparrow, Zonotrichia leucophrys. The song control nucleus HVC incorporates 50,000 new projection neurons into an existing pool of 100,000 neurons within one week of transitioning to breeding conditions. Within four days of transitioning back to nonbreeding conditions, an equal number of HVC neurons dies. Seasonal neuronal death in HVC increases neural stem cell proliferation in the adjacent ventral ventricular zone. This growth and regression correlates to respective increases and decreases in song production rate and quality. We found that microglia activation mediates pro-neurogenic responses in each of two paradigms: (i) inflammation induced locally with lipopolysaccharide microinjection in HVC was sufficient to induce neural stem cell proliferation; (ii) inflammation, including activation of microglia, was necessary to induce neural stem cell proliferation following seasonal neuronal loss. Having established that activation of microglia drives pro-neurogenic responses under non-pathological conditions, we are enabling future mechanistic studies with essential genetic and molecular tools, including a high quality annotated genome and transcriptome, cell-type specific molecular markers, in vivo imaging, and CRISPR/Cas9 mutagenesis.
CD34 count using Image based-immunofluorescence cell counter with filterpaper imbeded microchip
Authors: Lim Chae Seung , Jang Woong Sik , Nam Jeonghun
Published: January 2023

Background

The accurate measurment of CD34 positive cells is importance for successful hematopoietic stem cell transplantation. Until now, flowcytometric assay is the standard method for enumerating CD34+ stem cells it has some limitations. Microscanner C3(Biozentech, Korea) is noble image based cell counter for blood components adopt microscopy based cell counter with disposable plastic microchips. This system is composed of the staining solutions, the microscopic instrument and the image analysis software. We developed 0.5 um sized filter paper imbeded plastic microchip for enrichment of rare cells. and applied it to image based-immunofluorescence cell counter for CD34+ cells.

Methods

Leukocyte and CD34-positive cells were stained by CD45-BB515 and CD34-PE (Becton Dickinson, USA). We compared the reproducibility and linearity between the standard flowcytometric method (Beckman Coulter Cytomics FC500 ) and image based-immunofluorescence cell counter with filter paper imbeded microchip by using CD34 positive leukocyte quality control materials (300, 100, 50, 25,10 cells/μl).

Results

CD34 measurement by image based-immunofluorescence cell counter with filter paper imbeded microchip was performed within 5 min. The repeatability was within the range of 10–300 cells/μ, and the coefficient of variation was 8.2–20.3%. The correlation with the CD34 measured by flow cytometry was (R2> 0.9746, P <0.0001).

Conclusions

CD34 measurement by image based-immunofluorescence cell counter with filter paper imbeded plastic microchips is fast, simple and reliable, it could be used as simple measuring tools for rare blood cells in the blood.
Treatment with Multiple-linked Myelin Peptides Encapsulated within Nanoparticles Induces Antigen-specific Tolerance in SJL/J Relapsing-remitting Experimental Autoimmune Encephalomyelitis
Authors: Beddow Sara A , Neef Tobias , Ifergan Igal , Podojil Joseph R , Getts Daniel , Miller Stephen D
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a demyelinating disease of the central nervous system (CNS), and serves as a fit-for-purpose pre-clinical model of Multiple Sclerosis (MS). Data show that tolerogenic immune-modifying nanoparticles (TIMPs) encapsulating peptides/proteins is an effective therapeutic that induces antigen-specific tolerance to the encapsulated peptide/protein. The effectiveness of this therapeutic platform has been be demonstrated in multiple mouse models, as well as in a recently completed phase 2 double-blinded placebo-controlled clinical trial for the treatment of celiac disease. While previous EAE studies have utilized single peptides, the present study utilized a polypeptide containing the SJL/J mouse dominant encephalitogenic peptides (PLP139–151, PLP178–191, MBP84–104, and MOG92-10) linked together with intervening capsaicin S cleavage sites. The use of the multiple-linked myelin peptides was produced to achieve broader coverage of myelin-derived epitopes, which will be required for the treatment of MS. The present data show that this multiple-linked myelin peptide emulsified in CFA induced both CD4+ T cell responses and EAE in SJL/J mice similar to PLP139–151/CFA. Our data go on to show that treatment of SJL/J mice with multiple-linked myelin peptide TIMP inhibited both PLP139–151/CFA-induced R-EAE, as well as multiple-linked myelin peptide/CFA-induced EAE. Furthermore, treatment of SJL/J mice with multiple-linked myelin peptide TIMP significantly decreased TH17 cell responses and increased Tr1 cell responses. The present findings suggest that utilizing multiple-linked peptides may be clinically translatable for the treatment of autoimmune disease.
Farnesol reduces T cell infiltration in murine CNS inflammatory demyelination
Authors: Ramelow Christina C , Sell Lacey B. , Strawn Kevin D. , Hevrin Theresa , Kirby Trevor O. , Gibson K. Michael , Roullet Jean-Baptiste , Ochoa-Repáraz Javier
Published: January 2023

Farnesol (FOL) is a naturally-produced 15-carbon organic acyclic sesquiterpene alcohol (isoprenol) that acts as a potent blocker of neuronal voltage-gated Ca2+ channels (L- and N-type) and is found in the human brain. FOL has potent anti-oxidant and anti-inflammatory effects in vitro and is neuroprotective in a murine model of neurotoxicity. Because inflammation and neurodegeneration are mechanisms associated with CNS demyelinating diseases, we sought to determine whether FOL treatment would result in protection against experimental autoimmune encephalomyelitis (EAE). We compared the progression of EAE in MOG35–55 immunized female C57BL/6 mice treated orally with FOL (100 mg/kg/daily) emulsified in corn oil, versus vehicle-treated and untreated EAE mice. FOL significantly reduced the average clinical scores of EAE mice when compared to untreated mice and vehicle-treated mice. The protective effect was associated with a significant reduction of CD4+ T cell spinal cord infiltration in FOL-treated mice as assessed by flow cytometry. Although FOL’s mechanism of action remains to be known, we propose that FOL promotes protection against CNS inflammatory demyelination by promoting an anti-inflammatory effect. We compared pro-inflammatory and anti-inflammatory cytokine transcriptional levels in brain tissues of EAE mice and histological analysis of CD4+ T cell CNS infiltration and demyelination. The potential benefit of FOL nanoencapsulation in neuroprotection was explored. We demonstrate that there is a correlation between the reduced neuroinflammation and EAE severity observed in the context of FOL protection. Understanding the neuroprotective effects of FOL may provide insight to novel therapeutic approaches for MS.
Immunotherapy in Probable autoimmune encephalitis without detected autoantibody
Authors: Lee Woo-Jin , Lee Soon-Tae , Hong Sang Bin , Son Hyoshin , Jang Yoonhyuk , Sunwoo Jun-Sang , Park Kyung-Il , Chu Kon , Lee Sang Kun
Published: January 2023

Along with the rapidly expanding spectrum of autoimmune encephalitis (AE), probable AE with no detected autoantibody (AE-NoDab) has become a major category of AE. However, its clinical features, treatment, and prognosis were not demonstrated. In our institutional cohort of all consecutive patients with possible AE in 2012 – 2018, patients diagnosed with AE-NoDab were included in this study. AE-NoDab was subcategorized into limbic encephalitis (LE), acute disseminated encephalomyelitis (ADEM), and antibody negative probable AE (pAE). Patients’ clinical severity was assessed at every week for the first 12 weeks, at every month for the next 9 months, and then at every three months, using the modified Rankin scale (mRS) and the Clinical Assessment Scales in Autoimmune Encephalitis (CASE, score range 0–27) scores. Combination immunotherapy regimens used at each time point were categorized as steroid+immunoglobulin (SI), SI+Rituximab (anti CD-20 mAb, SIR), and SIR+ Tocilizumab (anti IL-6 mAb, SIRT). 136 patients (LE 52[38.2%], ADEM 14[10.3%], and pAE 70 [51.5%]) were followed-up for 39.0±19.6 (range 10–80) months. At last follow-up, 76 (55.9%) patients had favorable mRS outcomes (scores 0–2). Compared to 55 anti-NMDA receptor encephalitis patients, baseline severity was higher, use of immunotherapy was lower, and outcome was poorer in AE-NoDab. There was no difference in clinical characteristics, treatment, and outcomes among subcategories of AE-NoDab. In a linear mixed model analysis, using the SIRT regimen was more effective than conventional regimens such as SIR or SI in lowering CASE scores (both, P=0.001). Early diagnosis and use of combined immunotherapy consisting of SIRT is warranted to improve the poor outcomes of AE-NoDab.
Deficiency in B Cell Maturation Antigen reveals sex differences in Experimental Autoimmune Encephalomyelitis
Authors: Kumar Gaurav , Ko Rose M. , Axtell Robert C.
Published: January 2023

Multiple sclerosis (MS) is immune mediated demyelinating disease of the central nervous system (CNS) leading to neuronal damage. The disease is heterogeneous with respect to onset and clinical course, being more prevalent in women then in men with a ratio of 3:1. Further sex differences have been observed with males having a more severe and debilitating disease course compared to females. The complex interplay of immune cells, sex hormones or genetic differences could be contributing to these clinical phenomena, which remains to be elucidated. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, several reports have demonstrated no sex differences in disease onset or severity in C57BL/6 mice. Herein we report that deficiency in B Cell Maturation Antigen (BCMA) in C57BL/6 mice reveals a sex difference in EAE. BCMA plays a prominent role in B cell function, however the role BCMA plays in MS and EAE is unknown. Our initial goal of this project was to determine the impact BCMA deficiency has on EAE. In doing so we confirmed that EAE disease was not different in males and females from BCMA+/+mice. Strikingly in the BCMA−/− mice, there was a significant increase in EAE disease severity in males compared to females. The increased EAE in the BCMA−/− male mice was associated with increased demyelination and infiltration of inflammatory T cells and macrophages into CNS. Also, we observed decreased splenic transitional B cells in male BCMA−/− mice compared to females. Castration of mice significantly reduces EAE in BCMA−/− mice as compared to sham controls. Altogether, our data demonstrate that male hormones exacerbates EAE in BCMA−/− mice by augmenting inflammatory responses compared to females leading to increased neuro-inflammation.
Identification of markers of relapse in EAE using high-resolution quantitative mass spectrometry
Authors: Chase Carol Ann , Trementin Guillaume , Mullens Conor , Haskins William E , Forsthuber Thomas
Published: January 2023

Approximately 85 percent of individuals newly diagnosed with multiple sclerosis have the relapsing-remitting form of the disease characterized by attacks of neurologic symptoms that are unpredictable in occurrence and duration. Currently there are no clinically available biomarkers predictive of relapse. To address this need, we investigated CNS proteome changes over the disease course of relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in SJL mice as a preclinical model of the disease. Using a high-throughput quantitative preparation technique and high-resolution Bruker timsTOF mass spectrometry, we were able to identify thousands of unique proteins at each disease timepoint. Principal component analysis of protein expression confirmed that the remission and relapse phases of disease cluster independently, indicating distinguishable variation in protein expression profiles between the two disease states. Importantly, statistical testing identified proteins with differential expression in the CNS at different stages of disease, several of which are CNS specific. We are seeking to detect corollary changes in these CNS-specific proteins in the serum, pointing to a minimally invasive means of monitoring disease progress and measuring drug efficacy. Our study will validate homologous human biomarkers to guide treatment in individual patients and allow for proactive therapeutic intervention. Furthermore, our results may provide insights into mechanisms that contribute to disease pathology and offer novel therapeutic targets.
Case study: mepolizumab prevents progression of eosinophilic granulomatosis with polyangiitis with involvement of the central nervous system which is refractory to conventional immunotherapy
Authors: Son Hyoshin , Hong Sang Bin , Jang Yoonhyuk , Lee Woo-Jin , Sunwoo Jun-Sang , Jeon Daejong , Lee Soon-Tae , Park Kyung-Il , Lee Sang Kun , Chu Kon
Published: January 2023

Eosinophilic granulomatosis with polyangiitis (EGPA) is a systemic vasculitis involving small-sized vessel. It manifests mainly as asthma or eosinophilia, but the literature reported involvement of the central nervous system (CNS) in 5%, while isolated CNS involvement is very rare. We describe a case of isolated CNS involvement of EGPA which was refractory to conventional immunotherapy and treated with mepolizumab, an anti-interluekin-5 monoclonal antibody. A previously healthy 51-year-old man was referred to the neurology department for memory decline persisting for a month. Magnetic resonance imaging (MRI) showed the multifocal lesion in the periventricular and frontal lobe. Analysis of cerebrospinal fluid revealed an elevated protein concentration and leukocytosis. Considering his age, clinical course, and laboratory findings, steroid and intravenous immunoglobulin was started for autoimmune encephalopathy. Two months after discharge, the patient experienced new symptom – gait disturbance. The follow-up MRI showed increased extent of the previous lesions and the new lesions. To confirm the diagnosis, brain biopsy was done, and the finding was consistent with EGPA. We decided to start mepolizumab as second-line treatment, and observe the stationary course and absence of further progression of the lesion in the follow-up MRI. Mepolizumab for EGPA patients was introduced to achieve protocol-defined remission and clinical benefit compared to the placebo group in the several studies. Our case signifies possibility of mepolizumab to halt further progression of the disease clinically and radiologically, for the patient with isolated CNS involvement of EGPA which is unresponsiveness to conventional immunotherapy.
Cognitive deficit and neuroinflammation were abolished by a single adoptive transfer of Regulatory T cells in 3xTg mouse model of Alzheimer’s disease
Authors: Yang HyeJin , Park Seon-Young , Kim Kyunghwa , Baek Hyungjung , Hong Minwoo , Cha Nari , Bae Hyunsu
Published: January 2023

Alzheimer’s disease (AD) is characterized by progressive cognitive and functional loss leading to neurodegeneration. Although the details are unclear, recent substantial literature suggest that neuroinflammation can facilitate or initiate neurodegeneration of AD. We previously reported the therapeutic potential of regulatory T cells (Tregs) modulating neuroinflammation to protect cells against neurodegeneration. This study assessed the impact of regulatory T cells in the context of Alzheimer’s diseases and investigated the therapeutic effects by adoptive cell therapy with Tregs. When regulatory T cells were administered in the 3xTg animal model of AD, cognitive impairment was markedly improved. Microglial activation and reactive astrocyte in AD mice were dramatically abolished after a single injection of adoptive transfer of regulatory T cells. We confirmed these therapeutic potential of adoptive transfer of Tregs in intraventricular amyloid-β-injected mice model. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate neurodegeneration of Alzheimer’s disease (AD).
IFNAR signaling augments T cell and plasmacytoid dendritic cell activation during costimulation blockade resistant rejection
Authors: Habib Jakob , Mathews David , Dong Ying , Breeden Cynthia , Adams Andrew
Published: January 2023

Costimulation blockade (CoB) is a promising new transplant immunosuppression strategy offering improved long-term allograft survival without the nephrotoxicity of calcineurin inhibitors. However, increased risks of T cell mediated acute rejection have impeded the widespread adoption of CoB. Type I interferon (IFN) produced predominantly by plasmacytoid dendritic cells (pDCs) in response to innate immune signals, induce systemic inflammation that may prime the adaptive immune system for acute rejection. Here we investigate how signaling through the type I interferon receptor (IFNAR) contributes to CoB-resistant allograft rejection. We performed fully MHC-mismatched skin grafts from Balb/cJ donors to C57BL/6 recipients. Mice were treated with CoB alone (250 μg CTLA4-Ig + 250 μg anti-CD154 given by IP injection day 0, 2, 4, 6) or in conjunction with anti-IFNAR (250 μg given day 0, 2, 4, 6). CoB treatment improved graft survival but resulted in CoB-resistant rejection, while CoB+anti-IFNAR significantly improved graft survival. We found that CoB+anti-IFNAR led to a reduction in the activation and effector function of CD8+ T cells compared to CoB alone, demonstrated by reduced frequencies of CD44+CD8+ T cells and an inhibition of IFN-gamma and TNF-alpha production by CD8+ T cells. In addition, we observed reduced CD80 and MHC I expression on conventional DCs (cDCs) and pDCs treated with CoB+anti-IFNAR compared to treatment with CoB alone. These data suggest that signaling through IFNAR augments cDC and pDC activation and is associated with CD8+ T cell activation and effector functions in order to promote CoB-resistant allograft rejection.
Identification of immunologic factors associated with allograft rejection using NGS T cell receptor repertoire data
Authors: Vetter Julia , Aschauer Constantin , Heinzel Andreas , Reindl-Schweighofer Roman , Jelencsics Kira , Hu Karin , Oberbauer Rainer , Winkler Stephan , Schaller Susanne
Published: January 2023

T and B cells are known to play an important role in transplant rejection. Nevertheless, the factors that lead to rejection are not yet fully understood. We have developed a general bioinformatics pipeline for processing T cell receptor (TCR) and immunoglobulin (IG) repertoire next-generation sequencing (NGS) data for comparing immune repertoire properties between multiple groups of samples. Using such a pipeline can help to identify properties of the immune repertoire increasing the risk of allograft rejection after transplantation.

Our pipeline is implemented in Python 3.7. Various methods for processing, analyzing and comparing multiple immune repertoire NGS samples are provided. The pipeline returns detailed information about the sequencing quality and provides calculations and visualizations in regard of clonality, diversity, clonotype overlap as well as V(D)J gene analysis and similarity analysis of TCR and IG repertoires.

The functionality of the pipeline will be demonstrated on immune repertoire sequencing data from eight kidney transplant patients with sequential samples. For all patients mixed lymphocyte reactions (MLRs) have been performed to identify alloreactive T cells. With our clonotype overlap analysis module we were able to identify alloreactive clonotypes and to subsequently track changes in the alloreactive repertoire after transplantation.
Out-of-the-black-box: Standardisation of the EliSpot Method for immunomonitoring in personalized Immunosupression after solid organ tranplantation
Authors: Ettischer-Schmid Nicole , Strecker Kevin , Stein Maik , Reinke Petra , Preyer Rosemarie
Published: January 2023

Individual Immunotherapy for transplantation patients has been moved more and more into the focus of transplant centers, as individualized therapy approaches enable to minimize long-term side effects and increase the quality of life for the patients.

The Enzyme-Linked ImmunoSpot assay (EliSpot) has become a powerful tool in solid organ and stem cell transplantation. In an European multicenter study, the Bio-DrIM project c, the use of the EliSpot has been extensively evaluated with regard to monitoring of viral (Epstein-Barr Virus, EBV; Human Cytomegalovirus, HCMV and BK Virus, BKV) reactivation during immunosuppressive therapy after kidney transplantation.

The reference institute established SOPs and trained the attending sites. Additionally, EliSpot Reader Systems and EliSpot kits were extensively technical validated to guarantee that all centers reach the same results.

A total of 21 plates with 1983 wells were counted. Linear regression of all 1983 pairs of values lead to a linear regression line with the equation y=1.026*x+0,06888 and a Pearson correlation coefficient R2=9988, demonstrating the high concordance of each reader system.

To further demonstrate the extraordinary high standardization of the procedure, Inter- and Intra-Assay evaluation were performed. In the set of analyzed results the CV % varied under 20 %, which is far below cellular assays specified by the FDA with 25 %.

In conclusion, the combination of the EliSpot method with the EliSpot Reader System is a high-performance tool for diagnostics and monitoring of immunosuppression. Based on the high standardization potential, this state-of the-art technology will play an important role in diagnostic validation of next generation personalized therapy.
Regulation of ultraviolet radiation-induced cutaneous DNA damage by type I interferons
Authors: Sherwani Mohammad Asif , Ahmad Israr , Raman Chander , Elmets Craig , Yusuf Nabiha
Published: January 2023

Type I interferons (IFNs) are cytokines, that are important regulators of immune responses and are downregulated in human cancers, including skin cancer. Solar ultraviolet (UV) radiation is a proven environmental carcinogen, and exposure to solar radiation contributes to the high prevalence of skin cancer. The carcinogenic effects of UV light can be attributed to the formation of cyclobutane pyrimidine dimers (CPD) and errors in repair and replication of DNA. It is believed that type I IFNs reduce cellular proliferation and allow DNA repair in various diseases. Interferon receptor 1 (IFNAR1) is critical for signaling of type I IFNs. We hypothesized that type I IFNs (IFNα/β) will repair UVB induced DNA damage in mice. To test our hypothesis, shaved mice lacking IFNAR1 and wild type mice on a C57BL/6 background were irradiated with single dose of UVB radiation (100 mJ/cm2). Skin samples were examined for CPD by immunofluorescence and ELISA. We found that mice lacking IFNAR1 had more CPD as evident by ELISA and histology. In order to determine the cell type, which produces type I IFNs in skin after UVB exposure, single cell suspensions were prepared from irradiated skin samples and stained for type I IFNs using flow cytometry. We found that type I IFNs were produced by inflammatory monocytes (CD45+CD11b+Ly6C+). We also found that DNA repair gene XPA was downregulated in the mice lacking IFNAR1. In conclusion, our studies show that mice lacking the IFNAR1 had decreased repair of UVB induced CPD in the skin. Our ongoing studies will elucidate whether type I IFNs regulate development of UVB induced skin tumors.
Autocrine IL6 activates the STAT3-DNMT axis to silence the TNFa-RIP1 necroptosis pathway to sustain myeloid-derived suppressor cell survival and accumulation
Authors: Smith Alyssa D. , Lu Chunwan , Payne Daniela , Paschall Amy V , Klement John David , Redd Priscilla S , Ibrahim Mohammed , Yang Dafeng , Han Qimei , Liu Zhuoqi , Shi Huidong , Hartney Thomas , Nayak-Kapoor Asha , Liu Kebin
Published: January 2023

Accumulation of myeloid-derived suppressor cells (MDSCs) is a hallmark of cancer. However, the underlying mechanism of MDSC accumulation in the tumor microenvironment (TME) remain incompletely understood. We report that MDSC accumulation is regulated by the TNFα-RIP1-mediated necroptosis. We determined that inhibition of DNMTs with Decitabine (DAC) abolished MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes (CTLs) in tumor-bearing mice. DAC-induced decrease of MDSC accumulation is correlated with increased IRF8 expression in MDSCs. However, DAC also abolished MDSC-like cell accumulation in IRF8 KO mice, indicating that DNA methylation does not regulate MDSC lineage differentiation but mediates MDSC accumulation at post differentiation stage. We determined that DAC decreased MDSC accumulation through increasing cell death and identified RIP1-dependent necroptosis as target of DNA methylation in MDSCs. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter is hypermethylated in tumor-induced MDSCs in vivo. Consequently, DAC dramatically increased TNFα level in MDSCs and neutralizing TNFα significantly decreased MDSC cell death. Furthermore, recombinant TNFα induced MDSC cell death in a does- and RIP1-dependent manner. IL6 which is expressed in MDSCs in tumor-bearing mice and human colorectal cancer patients. Our data shows that the autocrine IL6 activates the STAT3-DNMT axis to epigenetically silence the TNFα-RIP1 necroptosis pathway to sustain MDSC survival and accumulation in cancer. Targeting the TNFα-RIP1 necroptosis is potentially an effective approach to supress MDSCs to activate tumor-reactive CTLs in the TME.
Hedgehog signaling induced metabolic reprogramming is essential for M2 polarization of macrophages in breast cancer
Authors: Hinshaw Dominique C , Hanna Ann , Sherpa Tshering , Samant Rajeev , Shevde Lalita
Published: January 2023

Current treatments for breast cancer offer little relief to patients with metastatic disease. The aberrant activation of Hedgehog (Hh) signaling enables cancer cells to survive, proliferate, and metastasize. Effective metastasis depends upon the surrounding immune microenvironment. Macrophages (MΦ) can be polarized to M1 (inflammatory) or M2 (suppressive), and increased populations of M2 tumor infiltrating MΦs are correlated with worse prognosis. We have discovered that Hh signaling contributes to M2 polarization. M1 and M2 MΦs have inherently different metabolic profiles, so we hypothesize that Hh signaling alters M2 MΦ metabolism to reflect M1 MΦ metabolism, leading to diminished M2 phenotype and function within the tumor microenvironment (TME).

Methodology includes a mouse model of mammary carcinoma, RNA-seq, mass spectrometry-based metabolomics, confocal imaging, and flow cytometry.

We elucidated the impact of Hh signaling blockade on the metabolism of MΦs diminishing their M2 polarization. Inhibition of Hh signaling reduces MΦ O-GlcNAcylation which directly correlates with the diminished suppressive attributes of M2 MΦs through the reduced activity of STAT6. Since STAT6 plays a dual role in governing the M2 phenotype and fatty acid oxidation, this ultimately enacts a metabolic cascade, including lipid utilization, cellular bioenergetics and mitochondrial morphology and function, that metabolically alters M2 MΦs to resemble M1 MΦs.

We have discovered that blockade of Hh signaling initiates a metabolic cascade diminishing the M2 phenotype and function. This knowledge will help us to better understand how to target and diminish the pro-tumorigenic phenotypes of MΦs in the TME and at the metastatic site.
Circular RNA AEBP2 modulates dendritic cells by the hnRNP/pP65 signaling pathway
Authors: Zhou Qinfeng , Wang Bowen , Koropatnick James , Min Weiping , McAlister Vivian , Greasley Adam , Zheng Xiufen
Published: January 2023

Circular RNA (circRNA) has been the focus of research in recent years. However, circRNA is rarely reported in dendritic cells (DCs). In our study, Microarray analysis of circRNA showed that circular RNA AEBP2 (circAEBP2) is significantly up-regulated in mature DCs compared with immature DCs. Knockdown of circAEBP2 in DC with siRNA arrests DCs at immature status with lower expression of MHC class II, CD40, CD80, and CD83, as well as decreasing the expression of Phosphorylation of nuclear factor-kappa B p65. CircAEBP2 -silenced DCs failed to activate allogeneic CD4+ T cells and CD8+ T cells in vitro. RNA immunoprecipitation assays show heterogeneous nuclear ribonucleoproteins (hnRNP) are enriched by circAEBP2 probes which have complementary circAEBP2 sequence, indicating circAEBP2 interacts with hnRNP. The effect of circAEBP2 silenced DCs on induction of anti-tumor immunity is investigated in an ongoing murine melanoma model. In conclusion, our results demonstrate that circAEBP2 regulates the development and function of DCs through interacting the hnRNP/pP65 signaling pathway. CircAEBP2 will be a new potential target for DCs-based immunotherapy.
PD-L1+PD-L2+ monocytes from patients with hepatocellular carcinoma may be associated with a poor prognosis
Authors: Yasuoka Hidetaka , Asai Akira , Matsui Masahiro , Okamoto Norio , Nishikawa Tomohiro , Ohama Hideko , Tsuchimoto Yusuke , Fukunishi Shinya , Higuchi Kazuhide
Published: January 2023

The prognosis of patients with hepatocellular carcinoma (HCC) is poor. Anti–programmed death-1 (PD-1) antibodies (Abs) has been approved to treat HCC. Some PD-1 ligands (PD-L1 and PD-L2) negative tumors respond to anti–PD-1 Abs. This response may be caused by the expression of PD-1 ligands on non-tumor cells. PD-1 ligands were recently found to be expressed on monocytes (CD14+ cells) from diverse cancer patients. In this study, we investigate the expression of PD-1 ligands on CD14+ cells from HCC patients and the role of CD14+ cells in an antitumor response.

From October 2017 to June 2019, 67 patients with HCC were enrolled. The expression of PD-L1 and PD-L2 on CD14+ cells was analyzed by flow cytometry. Their prognosis was evaluated by overall survival (OS). Tumoricidal activities of both CD14+ and CD8+ cells were calculated by LDH release assay.

In the results, CD14+ cells from the patients expressed PD-L1 (4.5–95.5%) and PD-L2 (1.6–90.2%). We set cut-off values as the mean values of PD-L1 (50.2%) and PD-L2 (50.5%) expression. In addition, according to the cut-off value, we classified patients as those either with PD-L1+PD-L2+CD14+ cells or other types of CD14+ cells. The OS of patients with PD-L1+PD-L2+CD14+ cells was significantly shorter than that of patients with other types of CD14+ cells (p = 0.0145). The tumoricidal activity of CD8+ cells from patients with PD-L1+PD-L2+CD14+ cells was suppressed by co-cultivation with syngeneic CD14+ cells. Furthermore, anti–PD-1 Ab restored the tumoricidal activity of CD8+ cells.

In conclusions, some patients with HCC have PD-L1+PD-L2+CD14+ cells that suppress their antitumor response of CD8+ cells. These inhibitory functions of CD14+ cells may be associated with a poor prognosis in such patients.
Adipocyte PD-L1 suppresses anti-tumor immune response and promotes breast cancer progression
Authors: Wu Bogang , Sun Xiujie , Gupta Harshita B. , Yuan Bin , Chiang Huai-Chin , Hu Yanfen , Curiel Tyler J , Li Rong
Published: January 2023

PD-L1 has become a major target in anti-cancer immunotherapy, however the overall response rate still remains relatively low among most types of cancers, notably breast cancer. There is an unmet need to boost efficacy of immune checkpoint blockade therapies. While both tumor and host immune cell-derived PD-L1 are implicated in immune suppressive functions of PD-L1, the exact immunosuppressive contribution of PD-L1 from different host tissues is little studied. Here we show that PD-L1 expression is significantly higher in human breast adipose tissue versus stromal vascular fractions. In vitro adipogenesis of mouse pre-adipocytes significantly up-regulates PD-L1 versus pre-adipocytes. In vitro co-culture shows that adipocyte PD-L1 suppresses T cell activation and response to anti-PD-L1. In an adipocyte-specific knockout (KO) mouse model, we show that syngeneic mammary tumors grow slower in KO than wildtype hosts. Immunophenotyping shows that tumors grown in KO mice have higher CD8+and CD4+ T cell infiltration as well as more CD8+ Prf+ cytotoxic T cells. Tumor tissue RNA-seq analysis reveals that genetic ablation of adipocyte PD-L1 confers a distinct transcriptomic signature of T cell activation and tumor killing. Our current findings uncover a previously unappreciated source of immune suppressive PD-L1 in the breast cancer microenvironment and could inform novel therapeutic strategies through targeting tumor-surrounding adipose tissue for treating breast cancer.
Intracranial tumors lead to sequestration of T cells in the bone marrow
Authors: Lorrey Selena J , Chongsathidkiet Pakawat , Wilkinson Daniel S , Champion Cosette , Fecci Peter
Published: January 2023

Glioblastoma (GBM) is the most common primary, malignant brain tumor and is universally fatal. Immunotherapeutic intervention has shown some success in immunogenic solid tumors, but little efficacy in GBM. Tumor-imposed T cell deficits remain a major obstacle. Recently, we demonstrated that GBM patients exhibit local and systemic T cell dysfunction, severe lymphopenia and contracted lymphoid organs. These observations are recapitulated in multiple murine models of GBM. Although T cell numbers are decreased in blood, lymph nodes, and spleen, we find large numbers of T cells sequestered in the bone marrow of both patients and mice with GBM. T cell sequestration occurs not only in the setting of GBM, but with nearly any cancer placed intracranially. Intriguingly, none of the tumors tested, including GBM, induce sequestration of T cells in the bone marrow when implanted subcutaneously. These data suggest that the capacity to sequester T cells resides within the brain and is usurped by tumors of the intracranial compartment. Such capacities may well represent a novel mechanism of immune privilege. Sequestration of T cells follows loss of sphingosine-1 phosphate-receptor 1 (S1P1) from the T cell surface. Understanding the drivers of S1P1 loss will provide crucial insight for how brain tumors facilitate systemic immune alterations and permit the development of more effective immune-based strategies for brain tumor patients.
A peptide presentation by an MHC tetramer reduces the expansion of the peptide-specific T cells and the reduced expansion is reversible by PD-1 blockades
Authors: Ouerkaxi Wushouer , George Marybeth , Daftarian Pirouz
Published: January 2023

Background

Peptide-MHC (pMHC) tetramers have been widely used in infectious disease and immune-oncology area to detect antigen-specific T cells. However, little is reported about their application in the context of pMHC presentation to stimulate T cells. Here we compared the expansion of antigen-specific T cells stimulated by an unlabeled pMHC tetramer and the peptide. We also analyzed the relationship among the programmed death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), PD-L1, CD80 and CD38 expression on antigen-specific and non-specific T cells.

Results

Compared to the peptide stimulation, the pMHC tetramer induced lower expansion of the peptide-specific T cells. PD-1, PD-L1, CD80, and LAG3 expression was significantly higher on peptide-specific T cells than on non-specific T cells. Interestingly, peptide-specific T cells expanded by pMHC tetramer induced lower numbers of dysfunctional PD-1+CD38high CD8+ T cells. Furthermore, blockade of the PD-1 pathway by using an FDA approved antibody reversed such a restriction of expansion.

Conclusion

The pMHC tetramer stimulation resulted in the reduced expansion of peptide-specific CD8 T cells when compared to the peptide stimulation. Since the impaired expansion was reversed by the anti-PD1 therapeutic antibody, this artificial stimulation method could be used as a functional in vitro immune-oncology drug screening tool.
Aluminum hydroxide and calcium phosphate adjuvants in adsorbed tetanus and diphtheria vaccines: Immunologic vs histopathologic effects
Authors: Bagriacik Emin Umit , Oruklu Nihan , Yaman Melek , Karakus Resul
Published: January 2023

We aimed to study effects of aluminum hydroxide (AH) and calcium phosphate (CAP) adjuvants in potentiation of new tetanus and diphtheria vaccines that have been developed in our laboratory. AH gel or CAP nanoparticles were prepared in vaccine formulations in adsorbed tetanus or tetanus-diphtheria (Td) vaccines. Vaccine formulations were injected to 8-week old female Balb/c mice. Only phosphate buffered saline (PBS) was injected to control animals. Tissue samples, peripheral blood samples and spleen cells were collected from each mouse. Serum was separated by centrifugation of peripheral blood. Spleen cells were stained with anti-CD4-FITC, anti-CD8-PE, and anti-CD19-APC monoclonal antibodies. ELISA was performed to test presence of anti-tetanus or anti-diphtheria antibodies. We found that either a single dose or an additional booster doses did not change significantly the composition of lymphocyte distribution in AH and CAP adjuvant injected mice in comparison to the control animals. No significant difference was detected for CD4/CD8 ratio and for CD19-expressing cell number. However, presence of IgM and IgG antibodies that were specific for both tetanus and diphtheria toxins increased significantly following injection of vaccines. Granulomas and cyst formation after subcutaneous injections of CAP-containing vaccines at the site of injection were evaluated. Supported by Gazi University BAP, No: 64/2018-04.
Single-Cycle Adenoviruses (SC-Ads) as Platforms for Immunostimulation
Authors: Barry Michael A , Barry Mary , Parrett Brian , Lu Shao-Chia , Zell Brady , Matchett William
Published: January 2023

Most adenovirus vaccines are replication-defective RD-Ads. In contrast, replication-competent adenovirus (RC-Ad) replicate antigen genes 10,000-fold in each cell to amplify immune responses. While RC-Ads vaccines are more potent than RD-Ad, they risk causing adenovirus infections in humans. We engineered “single-cycle” Ad (SC-Ad) vectors that still amplify transgenes, but that do not produce infectious progeny. RD-, SC-, and RC-Ads were tested by single immunization against influenza, Ebola virus, HIV-1, Zika virus, HCV, CMV, Clostridium difficile, and cancers in mice, hamsters, and macaques and antibody and T cell responses were measured. SC-Ad expressing GFP, influenza HA, or HIV gp140 generated antigens with 33 to 100-times less virus than RD-Ad. SC-Ad generated vaginal antibodies that persisted beyond 6 months after single intranasal immunization. Intramuscular injection of 1010 vp of SC-Ad6 expressing HIV-1 gp160 generated env antibodies in sera within 4 weeks of a single immunization in rhesus macaques. SC-Ad expressing a C. difficile toxin A and B immunogen generated toxin-binding antibody reciprocal titers above 105 that persisted in excess of a half of a year after a single immunization. Single immunization with SC-Ad protected vaccinated animals against toxin and against challenge with C. difficile spores. When SC-Ads were used to amplify the expression of immune stimulatory proteins in the context of cancer, single intratumoral injection of SC-Ad expressing 4-1BBL or GMCSF mediated significant reductions in tumor growth and extension of survival without the need for exogenous cancer antigen expression. These data suggest that SC-Ads may have utility against infectious diseases and cancer.