bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating nuclear organization of the inactive X in female lymphocytes
Authors: Sierra Isabel , Nguyen Son , Joyce Eric , Anguera Montserrat C.
Published: January 2023

Autoimmune diseases are devastating, incurable disorders that affect over 20 million people in the US, and many of these disorders have a striking female bias. The genetic basis for this sex-bias arises from the X chromosome. Female mammals, with two X chromosomes, utilize the epigenetic process of X Chromosome Inactivation (XCI), which transcriptionally silences one X. Expression of the long noncoding RNA Xist from the inactive X (Xi) reorganizes the Xi into a compact, bipartite structure, and targets the Xi to the nuclear periphery in somatic cells. The paradigm of XCI maintenance in somatic cells is stable association of Xist RNA and heterochromatic marks on the Xi, which function to maintain the Xi structure. Recently, we found a dynamic mechanism of XCI maintenance in female lymphocytes. In naïve female B cells, Xist RNA is not localized to the Xi, but after stimulation Xist RNA and heterochromatin marks return to the Xi. How this dynamic XCI process impacts Xi structure in lymphocytes is unknown, including which factors regulate Xi nuclear organization in this system. Using a novel allele-specific imaging system, we will present new findings on the compaction and organization of the Xi in lymphocytes. Additionally, we are investigating the regulatory roles of chromatin organization and nuclear structure proteins for lymphocyte-specific XCI maintenance, and how these factors regulate X-linked gene expression. Together, these results underscore the importance of Xi structure for the fidelity of XCI in female lymphocytes.
A germinal center B cell-specific long non-coding RNA regulates the germinal center response
Authors: Wikenheiser Daniel J , Weisel Florian , Chikina Maria , Shlomchik Mark J
Published: January 2023

Long non-coding RNAs are a unique class of molecules involved in an exceptional variety of cellular processes, including transcriptional, translational, and epigenetic regulation. Here, we report the initial characterization of a lncRNA expressed specifically in the germinal center--organized sites of B cell proliferation, somatic hyper-mutation, and cellular differentiation that develop in response to antigenic challenge. The precise signals dictating how and when differentiated cells, such as memory B cells and long-lived plasma cells, exit the GC reaction remain incompletely understood. Thus, additional levels of molecular regulation are likely at work in the coordination of these diverse processes. We identify GCLnc1--a novel, nuclear-localized lncRNA--as a regulator of the GC reaction. As GCLnc1 is located adjacent to the murine Bcl6 locus, this lncRNA has the potential to modulate expression of key transcription factors controlling B cell identity and/or differentiation. Interestingly, forced over-expression of GCLnc1 in B cells led to upregulation of TFs associated with plasma cell identity, such as IRF4 and Blimp-1. When adoptively transferred in vivo, over-expression of GCLnc1 led to transduced B cells predominantly adopting a non-GC phenotype at the typical peak GC response, in addition to enhancing expression of IRF4 and Blimp-1. Genetic deletion of GCLnc1 led to reduced frequency of GC B cells, decreased Bcl6 expression, and dysregulated light zone/dark zone distribution in response to NP-KLH immunization. Collectively, we suggest GCLnc1 plays a key role in the integration of signaling events during the GC reaction, and may modulate the processes that drive GC exit.
Downstream effectors of BCR signaling control transcription of autoimmunity-associated transcription factor Ets1
Authors: Kearly Alyssa , Saelee Prontip , Garrett-Sinha Lee Ann
Published: January 2023

The transcription factor (TF) Ets1 is a critical regulator of B cell quiescence. Its expression must be maintained at a high level in resting B cells to prevent inappropriate differentiation and autoimmune responses, and its downregulation in response to antigen receptor stimulation is vital for proper B cell responses. How Ets1 expression is controlled remains unstudied. Quantification of newly-transcribed Ets1 pre-mRNA shows that Ets1 gene transcription is reduced upon BCR stimulation. Inhibition of new protein synthesis did not prevent repression of Ets1 transcription in response to stimulation, suggesting that the phenomenon depends on modulation of existing TF binding to Ets1 regulatory elements (REs) rather than de novo synthesis of a repressor protein. Epigenetic analyses identified numerous putative REs in the mouse Ets1 locus. A 217 kb BAC transgenic construct containing potential RE sequences drives eGFP reporter expression in a manner that recapitulates that of endogenous Ets1. To identify REs that may be important for regulating Ets1 expression and stimulation-induced repression, we performed ATAC-seq in untreated and BCR-stimulated B cells, and observed potentially meaningful changes to chromatin accessibility. Motifs for highly relevant TFs are enriched in accessible regions in the Ets1 locus, including members of the Smad, ETS, AP-1, and NFκB families. Following up on NFκB as a potential regulator of Ets1 expression, downregulation of Ets1 transcription was found to be dependent upon IKK2 activity but did not require RelA. These analyses suggest a potentially complex regulatory network behind the expression of Ets1 as it safeguards the resting B cell state.

Funded by NIH R01 AI122720 and Lupus Research Alliance.
Robust serum-free expansion of human B cells with an animal component-free cell culture supplement
Authors: Antignano Frann , Babic Sandra , Eaves Allen C , Brown Mark A
Published: January 2023

Human B cells are utilized in workflows for discovering therapeutic antibodies and in the developing field of cellular therapy. The ability to expand the B cell population in blood samples from pathogen-exposed or naive patients offers the opportunity to improve the recovery of antibody sequences, increase the diversity of the B cell repertoire, or achieve de novo immunization in vitro. It can be difficult, however, to obtain robust expansion of B cells in cultures. Addition of serum improves performance, albeit with high lot-to-lot variability, and introduces the risk of contamination by adventitious agents. We have developed an animal component-free (ACF) supplement for culturing human B cells that does not require the use of serum, feeder cells, or specialized culture plates to achieve robust in vitro expansion. The ACF supplement is a 50X concentrate comprising non-animal-derived recombinant proteins and factors that can be added to a suitable base medium of choice to promote B cell activation and expansion. Human pan-B cells isolated from peripheral blood mononuclear cells (leukopaks) by immunomagnetic enrichment could be cultured and expanded in standard 24-well tissue culture plates for over 30 days, starting with seeding densities of 1 × 105 cells/well and passaging every few days from day 7 (± 1 day) onwards. Though pronounced inter-donor cell variability was observed in the rate of proliferation, an average ~55-fold, ~110-fold and ~160-fold expansion of viable B cells was obtained after 7, 11 and 14 days of culture, respectively (n = 21 donors; range 27- to 1090-fold at day 14 ± 1 day). Differentiation to plasma cells was apparent by flow cytometric analysis of CD138 and CD20.
Single cell transcriptomics of in vivo differentiating antibody-secreting cells reveals a divergent activated B cell program dependent on IRF4
Authors: Scharer Christopher , Patterson Dillon , Mi Tian , Price Madeline , Hicks Sakeenah L. , Boss Jeremy M
Published: January 2023

The generation of antibody-secreting cells (ASC) requires reprogramming the B cell transcriptome, epigenome, and metabolic potential to support the processes necessary to synthesize and secrete antibodies. These molecular changes have largely been studied at defined stages representing naive B cells, activated B cells, and ASC. To further refine when ASC fate decisions occur, we adoptively transferred CTV labeled B cells and monitored differentiation to both type I and II T cell independent antigens. At the peak of ASC responses, transferred cells were phenotyped by flow cytometry and isolated for single-cell RNA-seq. These data captured a continuum of responding B cells representing all stages of differentiation, with the largest heterogeneity observed among actB. Computationally ordering cells along differentiation trajectories, revealed divergent actB differentiation paths, with one branch leading to ASC formation that was dependent on IRF4. Moreover, cells that followed this trajectory downregulated the surface marker L-selectin and could be separated from cells that followed the alternative non-ASC differentiation branch. Indeed, isolation of actB by L-selectin status confirmed that L-selectin negative cells were ten times more likely to form ASC in culture. In summary these data provide insights into the cell division kinetics, molecular heterogeneity, and differentiation paths that lead to ASC formation.
Intrinsic Impairment of CD8 T Cells Induced by Helminth Coinfection
Authors: Rovira Eliezer , El-Naccache Darine W. , Zhao Yanlin , Gause William , Yap George S
Published: January 2023

Although it is known that helminth-triggered type 2 response increases host vulnerability to coinfections and potentially decreases vaccine efficacy, the exact mechanism(s) for the helminth-induced immunomodulation is still a fundamental question in the field. We have previously reported that CD44hiCD62L−KLRG1+ effector CD8 T cells from mice coinfected with Heligmosomoides polygyrus (H. polygyrus) and Toxoplasma gondii (T. gondii) exhibit a suppressed effector differentiation and impaired IFNγ production upon in vitro re-stimulation with toxoplasma antigens. This impairment was accompanied by a decrease in both, mitochondrial biomass and gene expression of the mitochondrial transcription factor, TFB1M. Further analysis of CD44hiT. gondii-specific CD8 T cells via single cell RNAseq, revealed an effector CD8 T cell subset from coinfected mouse that exhibit down-regulation of Cox17 and Atp5k transcripts. Both of these transcripts encode for proteins that belong to the electron transport chain multimeric complexes III and V, respectively. Taken altogether, our data suggest a potential metabolic-dependent mechanism of effector CD8 T cell insufficiency induced by helminth-coinfection. We hypothesize that helminth infection results in an impairment of CD8 T cell metabolic reprogramming, thus, affecting their effector development and function in response to T. gondii infection. The aim of this work is to increase the understanding of the helminth-associated immunomodulatory mechanism of CD8 T cells with the objective of finding therapeutic ways to restore CD8 T cells effector functions to improve vaccine efficacy in helminth-affected subpopulations.
Targeting CD49a+PBX1+ NK cells in recurrent spontaneous abortion immunotherapy
Authors: Yonggang Zhou , Binqing Fu , Haiming Wei
Published: January 2023

Objective

In the first trimester, CD49a+ natural killer (NK) cells are the main immune cells at the maternal-fetal interface. We have demonstrated that uterine NK cells have important physiological functions that can secrete various growth factors to promote fetal development. However, the functional subsets and transcriptional regulation mechanisms of NK cells expressing GPFs are unclear.

Result

Here, we have identified CD49a+PBX1+ NK cells as a unique subset in which the transcription factor, PBX1, upregulates the expression of the GPFs, pleiotrophin and osteoglycin, to promote fetal development at the maternal-fetal interface of both humans and mice. The results of ChIP experiments with anti-PBX1 antibody in decidual NK cells shown that PBX1 regulates the transcription of pleiotrophin and osteoglycin genes via direct binding. Inactivation of Pbx1 in mouse NK cells caused impaired fetal development due to a significantly decrease in the expression of GPFs. The results of exome sequencing of decidual NK cells from patients with unexplained recurrent spontaneous abortion (URSA), revealed decreased expression of PBX1 correlated with fetal growth restriction and pregnancy failure, suggesting that the impaired PBX1 gene in decidual NK cells is a potential biomarker for URSA.

Conclusion

Our study revealed that the transcriptional regulation mechanism in NK cells at the maternal-fetal interface, and suggested that impaired CD49a+PBX1+ NK cells contribute to the etiology of URSA, and provided the basis for improved approaches to NK cell-based immunotherapy for pregnancy related diseases.
Myeloid cells recruit colonic TCRab+ Intraepithelial Lymphocytes through Sphingosine-1-phosphate
Authors: Lefferts Adam R , Regner Emilie , Sansone-Poe Danielle , Norman Eric , Schultz Hannah , Kuhn Kristine
Published: January 2023

Intraepithelial Lymphocytes (IELs) are T-cells that exist in direct contact with the gut epithelium. IELs are important for barrier function; however, the specific pathway(s) by which they are recruited to the colonic epithelium remain unknown. To begin to address this question, we first performed a microarray comparing cell surface receptor expression in IELs versus lamina propria T cells. The array identified sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) as highly enriched in the IEL population. We also observed that tissue S1P levels are significantly lower in animals treated with antibiotics and in germ-free animals, both of which have reduced IEL numbers. Blockade of the S1P signaling axis during recolonization of an antibiotic-treated mice using FTY720 significantly reduced levels of TCRab+ IELs, but not TCRgd+ IELs. Next, since IEL recruitment has been shown to be Myd88 dependent, we used a series of lineage specific genetically deficient mice to identify the cellular source of the S1P. We found that LysM-Cre x Myd88fl/fl mice had significantly reduced TCRab+ IEL numbers in the colon. Additionally, Cre-negative but not LysM-Cre+ Myd88fl/fl deficient bone-marrow derived macrophages upregulate the primary kinase that generates S1P, sphk1, in response to stimulation with cecal contents, and induce T cell migration in a trans-well system in an S1P dependent manner. These findings indicate that TCRab+ IELs are recruited to the colonic epithelium by myeloid lineage cells via S1P in response to Myd88 mediated detection of microbial products. This is a new mechanism by which the microbiome influences colonic immunity, and indicates that TCRab+ IELs are dynamically recruited to the epithelium to mediate barrier function.
Th17 cell production of amphregulin contributes to the intestinal homeostasis
Authors: Zhao Xiaojing , Yang Wenjing , Yu Yanbo , Yu Yu , Zhou Zheng , Bilotta Anthony J , Yao Suxia , Cong Yingzi
Published: January 2023

Th17 cells have been implicated in the pathogenesis of inflammatory bowel diseases. Despite their inflammatory nature, Th17 cells are highly enriched in the intestines under homeostatic conditions. However, it remains unclear as to their exact role during intestinal homeostasis. We found that CBir1 TCR transgenic (CBir1 Tg) mice, which are specific for an immunodominant microbiota antigen CBir1 flagellin, demonstrated a higher levels of Th17 cells and higher amounts of amphiregulin (Areg), which has been recently shown to be highly protective at the mucosal surfaces, in the intestines compared to wild-type (WT) mice. Administration of Areg to WT mice led to decreased intestinal injury and higher level of tight-junction protein expression upon DSS insult. We further showed that Th17 cells produced higher levels of Areg compared to Th1 cells, and Areg inhibited T cell proliferation. Consistently, TCRβ/δ−/− mice transferred with Th17 cells developed less severe colitis compared with mice received Th1 cells. Additionally, TGF-β and IL-6, but not IL-23, the critical cytokines for Th17 development, promoted Areg expression in T cells in a time and dose dependent manner. Treatment with IL-21, but not IL-17, which are Th17 signature cytokines, upregulated Areg. Consistently, IL-21R−/− T cells produced lower levels of Areg. Mechanically, Stat3 mediates Areg expression in T cells, in that IL-6 promoted Stat3 activation, and IL-6 did not induce Areg expression in Stat3−/− T cells. Furthermore, Areg expression was lower in Stat3−/− T cells compared with WT T cells under Th17 polarization conditions. Collectively, these findings reveal that intestinal Th17 cells contribute to intestinal homeostasis and immune regulation by production of Areg.
Temporal changes in cellular sources of CXC chemokines control neutrophil recruitment during Citrobacter rodentium infection
Authors: Cai Baiyi , Zindl Carlene L. , Silberger Daniel J. , Figge David A. , Singer Jeffery R. , Merz Simon F , Gunzer Matthias , Weaver Casey T.
Published: January 2023

Enteropathogenic E. coli (EPEC) is one of the leading causes of diarrheal disease among children. Citrobacter rodentium (C.r) is a mouse model for EPEC and neutrophils have been found to be required for effective host protection against C.r. Yet details of the cellular sources that control neutrophil recruitment during C.r infection are poorly understood. Here we have found that C.r infection promotes neutrophil recruitment that is limited to distal colon where C.r colonizes. C.r infection induces the neutrophil-recruiting chemokines CXCL1, CXCL2 and CXCL5 in distal colon. Early in infection (D4) intestinal epithelial cells serve as the dominant source of CXCL1/2/5. However, later in infection (D8) neutrophils also provide CXCL1 and CXCL2, but fail to produce CXCL5, production of which is sustained in superficial epithelial cells. These findings support a model in which production of neutrophil-attracting chemokines is initiated in the infected epithelium and is amplified in a feedforward manner by incoming neutrophils. However, the unique production of CXCL5 by epithelial cells may be essential to recruit neutrophils to the mucosal surface, which C.r colonizes during infection.
Novel fluorochrome development enables 34-color high-content flow cytometry
Authors: Seong Yekyung , Nguyen Denny , Thakur Archana , Harding Fiona A , Nguyen Andrew
Published: January 2023

Flow cytometry is an essential technology to identify and quantify cell populations. Here we examined the development of new fluorochromes that could enhance flow cytometry’s capability. The latest spectral flow cytometers have 3 excitation lasers (405nm, 488nm, and 640nm) and incorporate the Avalanche Diodes Photodetector technology, which demonstrates significant improvement in sensitivity for the fluorescent emission signal longer than 800nm. However, there is no commercially available fluorochrome which could be excited by the above lasers and has peak emission signal beyond 800nm. To address the gap in technology, here we engineered 6 new fluorochromes: PE-750, PE-810, PE-830 for blue laser and APC-750, APC-810, APC-830 for red laser. These were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Then, the fluorochromes were conjugated with antibodies using Click chemistry, which is a simple, robust reaction with high-yield and high-reaction sensitivity. Also, we demonstrated long-term stability at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created a 34-color flow cytometry panel to measure broad human immune function with high sensitivity on a 3-laser Aurora. The additional UV laser upgrade will increase the instrument capacity to more than 40 color.

This panel will be applied to identify the expression patterns of many different receptors involved in trafficking on multiple different cell types from dissociated tumors and blood samples. In conclusion, this high-content flow cytometry panel will improve analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.
Demonstration of High Gain mode in combination with Imaging Flow Cytometry for improved EV analysis
Authors: Pugsley Haley Renee , Kong Raymond K
Published: January 2023

Extracellular vesicles (EVs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. In recent years, the importance of EVs has become apparent, as they are key mediators of intercellular communication. However, quantifying and characterizing EVs in a reproducible and reliable manner is challenging due to their small size—exosomes range from 30 to 100 nm in diameter. It is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize EVs using flow cytometric methods. However, these systems have not been designed to measure objects smaller than a cell. Here, we describe the use of High Gain mode on the Amnis® ImageStream® Imaging Flow Cytometer to address the challenges of measuring small particles. In this new High Gain mode, the charge-coupled device (CCD)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the EV. Object thresholds and masking have also been adjusted to better identify and detect small particles. Preliminary results using murine leukemia virus-sfGFP reference particles have shown up to a 5-fold increase in the number of GFP-positive objects collected in High Gain mode, when compared to standard gain on the ImageStream® System. In this study, we demonstrate improved small particle detection, including EVs, using this new High Gain mode.
Deep characterization of in vitro chronically stimulated T cells via single-cell multiomic analysis
Authors: Corselli Mirko , Saksena Suraj , Nakamoto Margaret , Lomas Woodrow E , Taylor Ian , Chattopadhyay Pratip K
Published: January 2023

To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. To comprehensively characterize in vitro chronically expanded human T cells, we performed single-cell multiomic analysis using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system for simultaneous measurement of the expression of 38 proteins and 399 genes. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states. T-cell activation induced by anti-CD3/CD28 beads and recombinant human IL-2 led to downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation, markers constitutively expressed throughout chronic activation, and markers uniquely up-regulated upon 14 days of stimulation. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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A novel radio-pharmaceutical to image activation of the innate immune system in vivo
Authors: Gammon Seth Thomas , Pisaneschi Federica , Paolillo Vincenzo , Qureshy Sarah , Piwnica-Worms David
Published: January 2023

Dysregulation of innate immunity contributes to the pathophysiology of many diseases, including obesity, rheumatoid arthritis and diabetes. Dysregulated production of reactive oxygen (ROS) and nitrogen species (RNS) by the innate immune system act both malevolently and benevolently through tissue damage and T-cell suppression. Optical reporters such as DCFDA and luminol/L-012 are routinely utilized to monitor ROS/RNS. 4-chloro-1-napthol (4CN) has been used to identify innate immune cells, and 1-napthol (N) covalently binds to activated human neutrophils through Nox2 and MPO-dependent mechanisms. However, these compounds are not suitable for deep tissue imaging in vivo. Under-standing this complex interplay in humans may benefit from deep tissue imaging of ROS/RNS by positron emission tomography (PET).

Herein we introduce [18F]4FN, a bioisostere of N and 4CN, as a novel PET imaging agent, and validate binding both in vitro and in vivo. In all-trans-retinoic acid-differentiated HL-60 cells, [18F]4FN showed a robust 4.7 +/− 0.5 fold increase in binding to PMA-activated cells vs control cells (SEM n = 4, p = 0.01). Binding was > 99% inhibited by treatment with the peroxidase inhibitor 4-ABAH and the Nox2 inhibitor DPI (p < 0.01, each). [18F]4FN was selectively trapped in inflamed mouse tissues in vivo upon activation by PMA or LPS, robust to injection route (ip vs iv) and strain (C57Bl6/N and Balbc/N). These data quantitatively co-correlated with a gold standard bioluminescent reporter, L-012 (r = 0.94, p = 0.0004), and immune infiltration was cross validated by H&E staining ex vivo. [18F]4FN showed strong promise in pre-clinical models and translational steps toward clinical PET imaging is underway.
Adarza’s ZIVA multiplex protein detection platform offers format for ultra-high sensitivity
Authors: Schmuke Jon J , Haley Jeaneen , Wente-Roth Traci
Published: January 2023

We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of both low and highly abundant targets. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Because of the label-free properties, sensitivity can be “fine-tuned” by leveraging a variety of mass enhancing options. We have previously demonstrated assays that respond in the nanogram to picogram/ml sensitivity using one format for enhancement. Herein, we demonstrate a newly developed mass enhancement option that increases sensitivity from very low pg/ml to mid-femtogram/ml levels. A custom designed biosensor with markers for inflammation and other custom needs was used to demonstrate this ultra-high sensitivity format in a multiplex array. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.
Protein- and sequencing-based massively parallel single-cell approaches to immune profiling asthma-specific cell subtypes and related gene expression biomarkers
Authors: Ji Xuhuai , Acharya Swati , Goncharov Igor , Lyu Shu-Chen , Miranda Molly , Padalon-Brauch Gilly , Nadeau Kari Christine , Maecker Holden T.
Published: January 2023

We implemented the BD Rhapsody Single-Cell Analysis System to assess cellular heterogeneity present in twins by capturing and sequencing over 40000 single cells across 10 samples from 5 twins patients with asthma or without asthma using oligos-conjugated antibodies and the whole blood immune targeted gene panel that utilizes multiplex PCR for detection of over 500 genes for immune cells subtyping, immunophenotyping, cell-type identification and markers correlated with stimulations. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We demonstrate the ability of the BD Rhapsody targeted panel to distinguish six different cell types and 18 distinct gene-expression biomarkers, including CLEC4E, CSF2, IL1RN, TPSAB1, CCL19, CCL22, CXCL2, LAMP3, SPP1, and VMO etc.. The HLA-DRA, HLA-DQB1, SPP1, and IL1RN had allelic polymorphism related to asthma. The BD Rhapsody targeted panel yields more sequencing depth with much fewer (<2%) sequencing reads per cell. The measurement of specific proteins and transcripts in individual cells is critical for understanding the role of cellular diversity in development, health, and disease.
Eukaryotic Translation Initiation Factor 4E (eIF4E) is Required for Development of T Follicular Helper Cells and Pathogenesis of Autoimmune Encephalitis
Authors: Patel Preeyam S , Walters Beth , Orlen Margo , Volkova Angelina , Ruggles Kelly , Park Christopher , Schneider Robert
Published: January 2023

Multiple sclerosis (MS) is a neuroinflammatory disorder resulting from infiltration of T cells into the central nervous system (CNS) and demyelination of neurons. Since T follicular helper (TFH) cells are associated with MS relapse, their selective inhibition could be an ideal therapeutic. TFH cells require transcription factor BCL6 and active mTORC1/2 for development. When mTORC is active, cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) initiates translation of selective mRNAs. However, the requirement of eIF4E for translation of mRNAs necessary for TFH cell development has not been investigated. Disruption of eIF4E binding to the 5′ mRNA cap with drug 4EGI-1 inhibits TFH and germinal center (GC) B cell development while having no effect on differentiation and effector function of TH1, TH2, TH17, or Tregs. Silencing of eIF4E in only CD4 T cells is sufficient to inhibit their formation of TFH cells. We used polysome profiling to determine which mRNAs are selectively translated by eIF4E and identified salient programs regulated by transcription (BCL6, NFAT) and costimulation (CD28, SLAM). eIF4E is required for translation of BCL6 in human lymph node TFH and GC B cells. Administration of 4EGI-1 during experimental autoimmune encephalitis (EAE) results in significantly decreased infiltration of CD4 T cells in the CNS, demyelination, and clinical score. Further, 4EGI-1 treatment following initiation of symptoms results in rapid improvement of symptoms and partial remission earlier than vehicle-treated animals. Thus, eIF4E is required for differentiation of TFH cells and pathogenesis of autoimmune encephalitis, and 4EGI-1 represents a potential therapeutic.
Schisandrin B and Alantolactone ameliorates experimental autoimmune encephalomyelitis in mice
Authors: Eken Ahmet , Cakir Mustafa , Karayigit Mehmet Onder , Altuntas Hamiyet Donmez
Published: January 2023

Schisandrin B (Sch-B) and Alantolactone (ALT) are two distinct molecules each of which could be extracted from herbs Schisandra chinensis and Inula helenium, respectively, commonly used in traditional Chinese medicine. Previous studies have revealed that both molecules impact STAT3, Nrf2 and Smad-mediated signaling pathways, and that Sch-B has protective effects in dextran sulfate sodium (DSS)-induced colitis models. However, besides a single paper studying Sch-B’s impact on murine Th17/Treg balance, the effects of Sch-B and ALT on human and murine Th17/Treg cells’ ontogeny and functions have not been investigated. In this study, we demonstrated that every other day administration of Sch-B or ALT during induction phase of myelin oligodendrocyte (MOG)35–55-induced experimental autoimmune encephalomyelitis (EAE) dramatically and significantly reduced EAE disease scores, demyelination, disease incidence, and infiltration of leukocytes into central nervous system, particularly IFN-γ+, IL-17+, GM-CSF+ T cells in C57BL/6 mice. Protection from disease correlated with elevated Treg and IL-10 levels. To our knowledge this is the first report demonstrating a prophylactic benefit for Sch B and ALT in a Multiple Sclerosis model and our results warrant further studies as to the use of both molecules after disease onset and mechanism of action.
A Phase 1 study comparing subcutaneous and intramuscular routes of administration of peginterferon beta-1a: macrophage-producing neopterin levels as biomarker
Authors: Butts Cherie L , Ramia Nancy , Zhao Yuan , Chen Kun
Published: January 2023

Background

Developing effective therapies for patients can be difficult as many fail due to safety concerns. Changes in route of administration (ROA) for approved therapies offers options to patients while mitigating known side effects. Peginterferon beta-1a (PEG) is approved for subcutaneous (SC) administration at 125 mcg every 2 weeks for patients with relapsing forms of multiple sclerosis. Intramuscular (IM) administration is associated with a different safety profile from SC and could offer patients receiving PEG an additional treatment option. To confirm efficacy is maintained, neopterin – a reliable pharmacodynamic measure for IFN therapies that is produced by macrophages – was tested in a bioequivalence study.

Methods

A two-period, crossover study with healthy volunteers who received a single dose of PEG administered SC followed by a single dose administered IM (or vice versa) was conducted. Blood samples were collected up to 504 hours post dose to evaluate pharmacokinetic and pharmacodynamic measures, including neopterin.

Results

The study enrolled 136 participants. Serum neopterin concentrations following SC and IM administration were similar, with maximal concentrations (Epeak) reached at a median ETmax of 40.1 hours and 44.0 hours, respectively. Geometric mean neopterin levels increased from baseline to maximum concentration similarly between both routes, with an increase from 8.0 to 22.6 nmol/L for SC and from 8.1 to 23.2 nmol/L for IM. The overall exposure to neopterin was also similar between both routes.

Conclusion

These results demonstrate no major difference in PEG activity; therefore, PEG administered SC or IM is expected to have a comparable pharmacological impact on the body.
Allo and autoimmune response following cardiac progenitor cells transplantation in rat myocardial infarction model
Authors: Gunasekaran Muthukumar , Mishra Rachana , Sharma Sudhish , Saha Progyapramita , Wang Lina , Davidson Lauren , Shah Aakash , Arfat Mir Yasir , Fu Xuebin , Abdullah Mohamed , Li Deqiang , Kaushal Sunjay
Published: January 2023

Cardiac progenitor cells (CPCs, c-kit+) are well-characterized stem cell type shown to potentiate cardiac regeneration in myocardial infarct (MI) model. However, auto and allo immune response to transplanted adult CPCs (aCPCs) reduces cell retention and MI recovery. We aimed to increase the regenerative potential of aCPCs by reducing immune response directed to the transplanted aCPCs using cyclosporine A (CSA).

Adult CPCs isolated from Wister Kyoto rat was transplanted into Brown Norway MI rats. MI rats transplanted with aCPCs+CSA demonstrated significant increase in MI recovery and cell retention compared to MI rats with aCPCs, CSA and Iscove Modified Dulbecco Media controls (p<0.05). MI rats transplanted with aCPCs showed increased infiltration of CD68+ cells and apoptotic cells in the infarcted myocardium compared to aCPCs+CSA group (p<0.05) analyzed by immunohistochemistry. Sera collected on day 2 and 7 of aCPCs group showed increased inflammatory cytokines (IL-2, IL-17, IFN-γ, TGF-β), antibodies to cardiac SAgs (Troponin-T and Myosin) allo antigens (RT1A, RT1B and RT1D) and reduced anti-inflammatory cytokines (IL-10). In contrast MI rats transplanted with aCPCs+CSA showed reduced inflammatory cytokines, abs to SAgs, allo antigens and increased anti-inflammatory cytokine IL-10 (p<0.05).

In conclusion, MI rats transplanted with allogeneic aCPCs with immune suppressant CSA showed increased cell retention, reduced inflammatory cells and cytokines compared to aCPCs, CSA and IMDM control. Therefore, CSA reduces auto and allo immune response and increases regeneration potential by increased transplanted aCPCs retention that resulted in increased myocardial recovery.
B cell-derived IL-1β and IL-6 drive homeostatic T cell recovery following lymphoablation
Authors: Hasgur Suheyla , Fan Ran , Valujskikh Anna
Published: January 2023

T cell reconstitution after lymphoablation in allograft recipients may lead to acute rejection and poor graft outcome. Using a mouse model of heart transplantation and murine anti-thymocyte globulin (mATG) we showed that while T cell recovery depends on B cells, B cell MHC expression is dispensable. The goal of this study was to investigate the role of B cell-derived IL-1β and IL-6 in T cell homeostatic reconstitution after mATG lymphoablation. The NanoString analysis showed that mATG upregulated IL-1β and IL-6 gene expression in recipient B cells. To validate NanoString findings, IL-1β and IL-6 expression was measured by intracellular flow cytometry. Following heart allograft placement and mATG treatment, B cells are the main source of IL-1β and IL-6, and these cytokines mutually regulate each other’s production. Either treatment with anti-IL-1β mAb or the use of IL-1R1 KO, caspase-1 KO and MyD88 KO recipients diminished both CD4+ and CD8+ T cell recovery along with improved heart allograft survival and decreased donor-reactive IFN-γ producing cells after mATG treatment. Similarly, in vivo IL-6 neutralization with mAb or using IL-6 KO recipients delayed CD4+ and CD8+ T cell recovery, markedly prolonged heart allograft survival and decreased donor-specific T cell responses suggesting that IL-1β and IL-6 are essential for optimal T cell recovery. Adoptive B cell transfers into μMT mice showed that B cell-derived IL-1β and IL-6 supported CD8+ T cells recovery. The absence of either IL-1R or IL-6R on CD8+ T cells markedly impaired their homeostatic recovery following mATG depletion. These findings reveal the essential role of B cell-derived IL-1β and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.
Novel insights into high alloantibody production in CCR5 KO recipient mice
Authors: Han Jing Light , Zimmerer Jason M , Zeng Qiang , Ringwald Bryce A , Cassol Clarissa , Warren Robert T , Abdel-Rasoul Mahmoud , Breuer Christopher K , Bumgardner Ginny L
Published: January 2023

Purpose

Conventional immunosuppressants do not effectively control development of humoral alloimmunity in a substantial proportion of kidney allograft recipients. CCR5 KO (H-2b) mice are a useful model to study antibody-mediated rejection: A/J (H-2a) kidney transplant (KT) recipients produce 10x more alloantibody (alloAb) versus wild-type (WT, H-2b) mice. CXCR5+IFNγ+CD8+ T (Tab-supp) cells are a novel regulatory CD8+ T cell subset that can kill alloprimed IgG+ B cells and mediate suppression of in vivo alloAb in an allogeneic hepatocellular transplant (HcT) model. We hypothesize that high alloAb-producing CCR5 KO KT mice have reduced CD8+ Tab-supp cells.

Methods

Investigate CD8+ Tab-supp cells in peripheral blood and splenocytes of CCR5 KO compared to WT HcT and KT recipients using flow cytometry.

Results

CCR5 KO compared to WT HcT recipients had fewer splenic Tab-supp cells (8.75±2.73×103 vs 19.1±3.07×103; p=0.03). CCR5 KO compared to WT KT mice produce higher alloAb by postoperative day (POD) 14 (5,800±700 vs 1,200±100; p=.0003) and have reduced peripheral CD8+ Tab-supp cells (650±400 vs 11,500±450 per 106 PBMC; p=0.002). Alloprimed CD8+ Tab-supp cells injected into CCR5 KO KT mice are detected in the peripheral blood, spleen, and lymph nodes, but not in the allograft. CCR5 KO KT mice that receive adoptive transfer of CD8+ Tab-supp cells on POD5 produce significantly less alloAb than WT KT mice (1,200±200 vs 5,800±700; titer assayed on POD 14; p<0.0001).

Conclusion

Deficiency of CD8+ Tab-supp cells is likely an important mechanism driving high alloAb production in CCR5 KO KT recipients. This subset exerts antibody suppressor function in lymphoid depots rather than in the kidney allograft.
DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival and Suppresses EGR1 and NFκB-mediated IL6 Production
Authors: Burdine Marie , Harrison David , Waldrip Zachary , Burdine Lyle
Published: January 2023

Organ transplantation is the only option for patients in end-stage organ failure. Therefore; it is imperative that immunological mechanisms that foster graft rejection be elucidated to develop more effective immunosuppressants for transplant patients.

In recent studies, the double-strand DNA break repair kinase, DNA-dependent protein kinase catalytic subunit(DNA-PKcs) has been implicated as a signaling molecule necessary for both B and T cell activity highlighting its potential role in graft rejection. In this study, we performed a phospho-proteomic screen to identify immunological targets of DNA-PKcs and a murine allogeneic skin graft study using DNA-PKcs inhibitors to investigate its effects on graft rejection. We identified a mechanism whereby DNA-PKcs is required for the protein stability of EGR1, a transcription factor known to promote NFκB expression. In primary splenocytes, DNA-PKcs inhibition abrogated EGR1 expression causing a decrease in NFκB-p65 levels and a reduction in the levels of the pro-inflammatory cytokine IL6 which can promote maturation of plasma cells. DNA-PKcs inhibition significantly reduced skin graft necrosis and extended graft survival compared to controls. DNA-PKcs inhibition reduced the infiltration of CD3+ lymphocytes into the grafts and the level of circulating CD19+ CD138+ lymphocytes. Additionally, DNA-PKcs inhibition reduced the production of donor-specific IgG and IgM antibodies. These results highlight a novel function for DNA-PKcs in the immune system and support further investigations into the use DNA-PKcs inhibitors in transplant therapy.
STING pathway mediated immunologic consequences of DNA damage repair gene losses in high-grade serous ovarian tumors
Authors: Shakfa Noor E. , Lightbody Elizabeth , Afriyie-Asante Afrakoma , Li Deyang , Chenard Stephen , Hamade Ali , Koti Madhuri
Published: January 2023

High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with high rates of chemotherapy resistance and poor outcomes. Our previous studies have demonstrated the variable tumor immune microenvironment states that associate with platinum chemotherapy response. We further showed the significance of the interferon induced chemokine CXCL10 as a key mediator of tumor infiltrating immune cell recruitment. Using the ID8-Trp53−/− murine model of HGSC, we demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC tumors to carboplatin chemotherapy and sensitizing them to immune checkpoint blockade. Notably, CXCL10 production via IFN1 is also governed by genes that regulate cellular DNA damage repair pathways. Evolving evidence indicates a role of BRCA1 and PTEN genes in mediating cellular IFN1 responses. Losses in the function of these genes is widely prevalent in a large proportion of HGSC tumors, where tumors with BRCA1 mutations have higher CD8+ T cell infiltration in contrast to those with loss of PTEN. We hypothesized that HGSC tumors with loss of PTEN expression can be rendered susceptible to immune mediated killing via activating the STING pathway. Tumors generated from ID8-Trp53−/−; Brca1−/− cells and those from ID8-Trp53−/−; Pten−/− cells in C57BL6 mice showed significant immunologic differences. STING agonist treatment significantly increased chemosensitivity and improved overall response in mice implanted with ID8-Trp53−/−; Pten−/− cells compared to those treated with carboplatin alone, altering immune responses. This study is foundational to guide rationalistic combinations of STING pathway activating therapies in HGSC.
CARD9 Induces Metabolic Reprogramming and Mediates the Immune Cascades in Dendritic Cell Against Pancreatic Cancer Progression
Authors: Lu Yi , Tian Chen , Gou Jing , Xiang Ming
Published: January 2023

Caspase recruitment domain family member 9 (CARD9), a signaling adaptor protein, transduces the immune signals from pattern recognition receptors (PRR) and induces the cascaded immune response, which plays a critical role in innate and adaptive immunity. In order to explore the role of CARD9 in DCs during pancreatic cancer (PC) progression and its underlying mechanism, samples of PC in patients with different clinical stages, animal model of PC, simulated immune microenvironment in vitro were employed. We found that the immunogenicity of bone marrow-derived DCs decreased in the progression of PC, which was accompanied with downregulated CARD9 expression. And the glycolysis in DCs was attenuated accompanied with diminishing glucose uptake and reducing acidic metabolites production after CARD9 knockdown, while fatty acid oxidation (FAO) was lifted. Further, CARD9 deficiency weakened the DCs migration, antigen presentation and stimulation to CD8+ T cell immune response. Mechanically, activated CARD9 recruited Bcl10 and MALT1 and constructed CARD9-Bcl10-MALT1 signalosomes, followed by the PI3K/AKT/mTOR pathway mediated metabolic switching and NF-κB pathway induced inflammatory cascades. Furthermore, DCs with CARD9 overexpression loading antigenic peptide mucin 1 (MUC1) curbed tumor growth in mice with PC subcutaneously implanted tumor. These results reveal a new role of CARD9 as a molecular switch of glycolysis in DCs that triggers the metabolic reprogramming, antigen presentation and antitumor effects of CD8+ T cells.
Immune landscape of genetically-engineered murine models of glioma relative to human glioma by single-cell sequencing
Authors: Zamler Daniel Bernard , Shingu Takashi , Heimberger Amy , Hu Jian , Draetta Giulio F.
Published: January 2023

Glioblastoma (GBM) is the most frequent and deadliest primary brain tumor, with a <10% 5-year overall survival rate. Despite promising results in other disease contexts, deployment of immune system-targeted therapies against GBM has not been successful thus far. In this regard, few preclinical models effectively recapitulate the human GBM immune microenvironment, hindering our ability to identify potentially targetable vulnerabilities. In this study, we characterized tumor immune microenvironment of the Qk/trp53/Pten (QPP) triple-knockout mouse model, harboring alterations common in human GBM and found that tumor infiltrates contain a predominantly myeloid cell population of monocytes, macrophages, and resting dendritic cells, with minor populations of T, B, and NK cells. Notable differences between spontaneous QPP tumors and tumors derived by implanting established QPP cell lines included T-cell enrichment and a larger proportion of myeloid-derived suppressive cells (MDSCs) in implanted tumors. Profiles of myeloid cells subtypes in QPP tumors paralleled findings in human GBMs, suggesting that this model effectively recapitulates the complexity of the myeloid-cell compartment and other human GBM immune cell populations.