bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel multicolor stimulated emission depletion (STED) microscopy strategy to visualize mitochondrial morphology in lymphocytes
Authors: Roesler Alexander Scott , Miozzo Pietro , Smelkinson Margery , Akkaya Billur , Brzostowski Joseph , Kabat Juraj , Theall Brandon P. , Traba Javier , Pierce Susan K. , Akkaya Munir 
Published: January 2023

Abstract
Immunometabolism is an emerging field that studies the activation-induced reprogramming required to alter immune cell function and meet new energetic demands. In-depth investigations of the metabolism-related cellular changes necessitate high-resolution visualization of mitochondria. However, current strategies for imaging mitochondria are optimized for model cells with high cytoplasm-to-nucleus ratios and are not readily adaptable for many immune cells. Here, we devised a multicolor STED microscopy method to assess B lymphocyte mitochondria in resting and activated states. By staining specimens with TOM-20 and MitoTracker dyes, as well as DAPI and a viability dye, our strategy allowed us to assess the morphology and volumes of mitochondria while efficiently excluding dead cells. Our analyses showed that stimulation of B lymphocytes via toll-like receptor 9 (TLR9) and/or B cell receptor (BCR) led to notable mass and volume increases in individual mitochondria within 24 hours. Furthermore, TLR9-stimulated cells demonstrated more tubular and connected mitochondrial networks consistent with their higher metabolic demands. In addition, collecting images of the same specimens side-by-side on AiryScan and standard confocal laser microscopes showed that STED microscopy performed significantly better in resolving changes in 3D mitochondrial morphology. However, none of the light microscopy imaging strategies could resolve activation-induced structural alterations in cristae or mitochondrial matrix. In conclusion, our novel STED-based multicolor imaging approach offers a powerful super-resolution tool to study immunometabolism and mitochondrial reorganization in lymphocytes at the whole cell level.
T lymphocyte immunophenotyping: 13-color flow cytometry panel design using the NovoCyte flow cytometer
Authors: Jachimowicz Lauren A. , Ye Peifang , Guenther Garret , Abassi Yama 
Published: January 2023

Abstract
The human immune system is highly complex and immune status is associated with disease status, treatment efficiency, and response to external stimuli such as vaccines. For example, the immunophenotyping of leukemia has become indispensable for diagnosis of hematological malignancies. Monitoring T lymphocytes has gained diagnostic importance in the prognosis and prediction of therapies. In this study, a series of T-cell markers previously used in the HIPC (Human Immunology Phenotyping Consortium) project were assessed. Through comprehensive consideration and analysis of the expression of each marker, fluorescence intensity, spectral overlap, and NovoCyte configuration, a 13-color T-cell phenotyping panel was designed. Next, peripheral blood mononuclear cells (PBMCs) were tested on a NovoCyte. Live T cells (CD3+/Aqua−) were identified as either T helper cells (Th) or cytotoxic T cells (Tc) cells by expression of CD4 and CD8 antigens. Next, further T cell subtype was determined by CCR7 and CD45RA: naïve (CD45RA+/CCR7+), effector (TEF, CD45RA+/CCR7−), effector memory (TEM, CD45RA−/CCR7−) and central memory (TCM, CD45RA−/CCR7+). Activation of T cells was assessed by the activation marker HLA-DR. Identification of Th subtypes (Th1, Th2 and Th17) was determined by CXCR3 and CCR6. Regulatory T cells (Treg) were identified as CD4+/CD25+/CD127lo/CCR4+, and memory and naïve Treg cells were further differentiated by CD45RO. With this panel, the phenotype and activation of T cell subtypes can be determined in one run which allows quickly and in-depth analysis of human T lymphocyte subsets. NovoCyte flow cytometer features high resolution of dim signals and flexible configuration and is particularly suitable for multicolor analysis.
RTA 1701 is an oral RORγt inhibitor that suppresses the IL-17A response in non-human primates
Authors: Reisman Scott A. , Lee Chun-Yue I. , Proksch Joel W. , Sakamoto Mitsumasa , Ward Keith W. 
Published: January 2023

Abstract
RTA 1701 is an orally-bioavailable, selective RORγt inhibitor. RORγt orchestrates the differentiation of Th17 cells and contributes to autoimmune disease pathogenesis. Inhibition of RORγt suppresses Th17 cells and the associated secretion of the effector pro-inflammatory cytokine IL-17A. As such, RTA 1701 is highly efficacious in rodent models of rheumatoid arthritis and multiple sclerosis. This study was conducted to evaluate the pharmacokinetics of RTA 1701 and its effects on ex vivo stimulation of IL-17A production in whole blood after oral administration to naïve healthy cynomolgus monkeys. Monkeys received a single administration of RTA 1701 (0.3, 3, 30, or 300 mg/kg). After a one-week washout, monkeys received the same doses of RTA 1701 once daily for 14 consecutive days. Blood was collected after a single dose and after 14 days of dosing for analysis of plasma RTA 1701 concentrations through 72 hours post-dose and for evaluation of RTA 1701 effects on ex vivo stimulation of IL-17A at 24, 30, and 72 hours post-dose. RTA 1701 exhibited oral bioavailability in monkeys with dose-dependent increases in exposure over the wide range of doses evaluated. Further, oral administration produced systemic exposure to RTA 1701 at sufficient levels to produce significant and dose-dependent suppression of ex vivo stimulation of IL-17A secretion in whole blood at 24 and 30 hours after single and repeat dosing, with IL-17A levels returning to baseline within 72 hours after a single dose. Collectively, these data demonstrate that RTA 1701 is orally bioavailable in non-human primates and significantly suppresses the IL-17A response. These data also support the future clinical development of RTA 1701 for the potential treatment of autoimmune diseases.
Targeting Dendritic Cells with Alpha 1 Antitrypsin has Therapeutic Potentials in Lupus
Authors: Elshikha Ahmed Samir , Yuan Ye , Lu Yuanqing , Chen Mong-Jen , Akbar Mohammad A. , Plate Henrike , Zeumer Leilani , Morel Laurence , Song Sihong 
Published: January 2023

Abstract
Increasing evidence indicate that dendritic cells (DCs) play a critical role in the development and pathogenesis of systemic lupus erythematosus (SLE). Targeting DCs in lupus may be beneficial, but it has not been yet achieved effectively. Alpha 1 antitrypsin (AAT) is a multifunctional protein with anti-inflammatory activity and immunoregulatory functions. In this study, we tested the effects of AAT on DCs and B cells in vitro and its therapeutic potential in vivo in two spontaneous mouse models of lupus. We demonstrated that AAT treatment significantly inhibited DC maturation, induced from C57BL/6 or B6.Sle1.Sle2.Sle3 lupus-prone mice, (using MHC-II IA, CD80, CD86 and CD40 as markers) and function (IFN-I, IL-1β, TNF-α, and IL-12 secretion) in response to TLR4 and TLR9 stimulation. Interestingly, the inhibitory effect of AAT on DCs inhibited B cell proliferation and antibody production induced in DC – B cell co-cultures. Importantly, in vivo AAT treatment prevented the development of kidney disease in MRL/lpr mice and AAT treatment delivered with an AAV-gene therapy vector extended the life span of NZM2410 mice, with 70% mice that received AAT remaining alive at 72 weeks of age vs. all control animals died by 46 weeks of age. In all studies, AAT treatment significantly reduced lupus-associated autoantibody levels, including anti-dsDNA IgG and anti-nuclear autoantibodies. Considering the well-known safety profile of AAT, our results strongly suggest a therapeutic potential of AAT for the treatment of SLE in humans.
This work was supported by grants from University of Florida Research Foundation and Grifols Inc.
Induction of Haploidentical Mixed Chimerism Cures Autoimmune Type 1 Diabetes
Authors: Liu Yuqing , Zhang Mingfeng , Riggs Arthur , Zhang Xi , Zeng Defu 
Published: January 2023

Abstract
Type 1 diabetes is an autoimmune disease in association with particular MHC, and induction of MHC-matched mixed chimerism cannot reverse autoimmunity. Haploidentical HCT has gradually become a common practice for treating non-malignant diseases, but it remains unknown whether induction of haploidentical mixed chimerism can reverse autoimmunity.
In the current studies, we induced mixed chimerism in NOD mice (H2-Ag7) by transplanting bone marrow and CD4+ T-depleted spleen cells from haploidentical (B6 × B6g7) F1 donors (H2-Ab/g7). We found that haploidentical mixed chimerism eliminated insulitis as well as prevented in pre-diabetic and cured diabetes in new-onset WT NOD mice. Interestingly, although I-As was thought to be autoimmune susceptible, induction of haploidentical mixed chimerism with I-Ag7/s transplants also effectively eliminate insulitis. Furthermore, the mixed chimerism markedly reduced the percentage of host-type but not donor-type CD4+CD8+ thymocytes and increased CD11c+CD8−SIRPα+CD11b+ migratory DC that can mediate thymic negative selection in the chimeric thymus. In the peripheral, percentage of tetramer+ autoreactive host-type Tcon cells was markedly reduced and part of the residual T cells became PD-1hiIL-7Rαlo anergic/exhausted, while percentage of host-type CD4+Foxp3+ Treg cells significantly increased. Additionally, in the pancreas of mixed chimeric BDC2.5 NOD mice, Treg cells among tetramer+ autoreactive T cells increase 5–10 fold. Taken collectively, haploidentical mixed chimerism reverses autoimmunity and eliminating insulitis in NOD mice by enhancing DC mediated negative selection in thymus and augmenting expansion of Treg, especially antigen specific Treg cells, in the periphery.
Discovery of a novel human anti-IL-2 antibody that potentiates Regulatory T cells by a structure-based mechanism
Authors: Trotta Eleonora , Bessette Paul H. , Silveria Stephanie L. , Ely Lauren K. , Jude Kevin , Holst Charles R. , Coyle Anthony , Garcia Christopher K. , Crellin Natasha K. , Rondon Isaac J. , Bluestone Jeffrey A. 
Published: January 2023

Abstract
Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Treg). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Treg cells. We report the generation of the first fully human anti-IL-2 antibody, F5111.2 that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalitis, and protected mice against xenogeneic Graft-versus-Host-Disease (GvHD). These results suggest that IL-2-F5111.2 may provide a new immunotherapy to treat autoimmune diseases and GvHD.
APD588, a novel, selective S1P- receptor modulator, inhibits inflammation and airway hyperresponsiveness in the ovalbumin-induced model of allergic asthma
Authors: Crosby Catherine Marie , Komori H. Kiyomi , Christopher Ronald , Adams John W 
Published: January 2023

Abstract

Background
FTY720 is a non-selective sphingosine-1-phosphate (S1P) receptor modulator that has shown efficacy in mouse asthma models. APD588 is an investigational stage S1P receptor modulator with increased selectivity and potential for an improved safety profile. We hypothesized that the optimized S1P receptor profile of APD588 would confer beneficial effects in ovalbumin (OVA)-induced asthma.

Aim
To evaluate efficacy of prophylactic APD588 in the OVA-induced allergic asthma model

Methods
Receptor potency and selectivity profile of APD588 was determined (b-arrestin, GTPgS) in cell lines expressing recombinant human S1P1–5. Efficacy in allergic asthma was assessed in BALB/c mice which were sensitized with intraperitoneal OVA on days 0, 7, and 17, then exposed to intratracheal (IT) vehicle or OVA on days 21–23. From day 20, vehicle, APD588 or FTY720 were dosed IT, QD. On day 24, methacholine-induced airway hyperresponsiveness (AHR), immune cell infiltration and BAL cytokines were measured.

Results
APD588 was a potent modulator of S1P1, with less potency at S1P4, 5, and no detectable activity at S1P2,3. IT administration of APD588 produced a dose-dependent inhibition of AHR, significantly inhibited airway eosinophil and lymphocyte infiltration, and BAL cytokines IL-4 and IL-5. FTY720 also reduced AHR and neutrophil and lymphocyte BAL cell counts, but had no effect on eosinophil counts or cytokines.

Conclusions
APD588 was an effective IT therapy for OVA-induced asthma. Lung function improvements correlated with reduced Th2 cytokines and lymphocyte and eosinophil infiltration in the BAL. Based on these results, APD588 may have therapeutic potential in asthma, and broadly indicate potential in other atopic Th2 diseases.
Sox9 deletion in the intestinal epithelium increases house dust mite induced allergic asthma symptoms in mice
Authors: Joldrichsen Marisa R , Kim Eunsoo , Attia Zayed , Davis Ian C , Comet-Boyaka Estelle , Boyaka Prosper N 
Published: January 2023

Abstract
Paneth cells regulate many key aspects of gastrointestinal health through the antimicrobial products and cytokines they produce. Loss or defective Paneth cell functions leads to dysbiosis and is one of the causes of Inflammatory Bowel Disease (IBD). Mice with Sox9 gene deletion within the intestinal epithelium (Sox9 ΔIEC mice) lack Paneth cells making these mice a great model for the study or IBD and other consequences of dysbiosis. Our separate studies have shown that dysbiosis of the gut microbiota could enhance allergic sensitization in the gut and subsequent allergic responses to nasal allergen challenge. We investigated whether dysbiosis caused by a Sox9 deletion in the intestinal epithelium of mice could affect allergic sensitization in the airway and development of asthma responses. For this purpose, control wild-type and Sox9 ΔIEC mice were sensitized tohouse dust mite (HDM) by daily intranasal administration over two weeks period. Upon nasal HDM challenge, Sox9 ΔIEC mice showed more severe signs of allergic asthma than wild-type C57 BL/6 mice as indicated by their higher hypothermia. We also established that the increased allergic symptoms in Sox9 ΔIEC mice correlated with higherIgE levels in serum and bronchoalveolar lavage fluid, and higher frequency of HDM-specific Th2 cells but no difference in Th1 or Th17 responses. These results further illustrate that the gut immune homeostasis plays an important role in allergic responses at distant mucosal sites.
Release of transglutaminase 2 from respiratory syncytial virus-infected airway epithelial cells and its role in disease pathogenesis
Authors: Hosakote Yashoda Madaiah , Rayavara Kempaiah , Kurosky Alexander 
Published: January 2023

Abstract
Respiratory syncytial virus (RSV) is the common cause of severe lower respiratory tract infections in young children, the elderly and the immunocompromised, for which no effective treatment or vaccine is currently available. RSV infections have also been linked to both the development and severity of asthma. Oxidative stress plays a major role in the pathogenesis of many lung inflammatory diseases. Our studies have shown that oxidative stress responses in the airways play a major role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium levels, which in turn activate calcium dependent enzymes, including transglutaminase 2 (TG2). TG2 is a transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications and has been implicated in the pathogenesis of many diseases. This study’s objective was to investigate the release of TG2 in airway epithelial cells in response to RSV infection and its potential mechanism. Studies were conducted in airway epithelial cells, a human lung carcinoma cell line and normal small alveolar epithelial cells. The changes in the TG2 levels were measured by Western blot and RT-PCR after cells were pretreated with antioxidants and infected with RSV. TG2 levels were significantly increased in the secretome with a concomitant decrease in the cell lysates, cytoplasmic and nuclear extracts in response to RSV infection. Antioxidants treatment significantly inhibited RSV-induced TG2 release. TG2 release was induced only by RSV but not RSV-induced cytokines or chemokines. These results suggest that modulation of oxidative stress represents a potential novel pharmacological approach to ameliorate virus-induced acute airway disease through TG2 release blockade.
T follicular helper cell recruitment to the ischemic brain
Authors: Kissel Lee T , Rayasam Aditya , Kijak Julie , Choi Yun Hwa , Hsu Martin , Kovacs Krizstian , Sandor Matyas , Fabry Zsuzsanna 
Published: January 2023

Abstract
Stroke is one of the leading causes of death and disability worldwide. Both clinical and preclinical studies suggest the importance of inflammation in acute and long-term neuronal tissue damage following ischemic stroke. However, the mechanisms of how components of the adaptive immune system contribute to neuroinflammation after stroke are still not fully understood. Previous work in our lab demonstrated that CD4+ T cells accumulate in the brain after cerebral ischemia and Interleukin-21 (IL-21) plays a significant role in T cell-mediated reperfusion damage following ischemic stroke. Given that the major producer of IL-21 is follicular helper T (TFH) cells, we sought to delineate the contribution of these IL-21 producing cells to ischemic damage. Here, we identify that pro-inflammatory CD4+CXCR5+IL21+ TFH cells are recruited to the ischemic brain during the reperfusion phase of transient middle cerebral artery occlusion (tMCAO) in mice. We also show that in human stroke tissue, CD4+CXCR5+ cells adhere to CXCL13-expressing inflamed vessel within the stroke lesions. Additionally, we found that inflamed endothelial cells in mice upregulate CXCL13 following tMCAO, indicating a possible mechanism contributing to TFH cell recruitment to the ischemic brain. Most importantly, administration of blocking antibodies against CXCL13 or BCL6, a TFH cell transcription factor, after tMCAO resulted in a significant decrease in infarct volume and TFH cell frequency in the brain. Overall, these data identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and identify potential novel targets for stroke therapy.
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer
Authors: Lio Chan-Wang Jerry , Shukla Vipul , Samaniego-Castruita Daniela , Avalos Edahi Gonzalez , Chakraborty Abhijit , Yue Xiaojing , Schatz David G , Rao Anjana 
Published: January 2023

Abstract
TET enzymes are dioxygenases that promote DNA demethylation by oxidizing the methyl group of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here we report a close correspondence between 5hmC-marked regions, chromatin accessibility and enhancer activity in B cells, and a strong enrichment for consensus binding motifs for basic region-leucine zipper (bZIP) transcription factors at TET-responsive genomic regions. Functionally, Tet2 and Tet3 regulate class switch recombination (CSR) in murine B cells by enhancing expression of Aicda, encoding the cytidine deaminase AID essential for CSR. TET enzymes deposit 5hmC, demethylate and maintain chromatin accessibility at two TET-responsive elements, TetE1 and TetE2, located within a superenhancer in the Aicda locus. Our data identify BATF as a key bZIP transcription factor involved in TET-dependent Aicda expression. 5hmC is not deposited at TetE1 in activated Batf-deficient B cells, indicating that BATF facilitates TET recruitment to this Aicda enhancer. Our study emphasizes the importance of TET enzymes for bolstering AID expression, and highlight 5hmC as an epigenetic mark that captures enhancer dynamics during cell activation.
Direct activation of Toll-like receptors induce differential B cell responses
Authors: Nelson Alexander J , Wu Yee Ling 
Published: January 2023

Abstract
B cells coordinate host defense against microbes by producing antibodies, secreting cytokines, and presenting antigens to T cells. Recognition of pathogen-associated molecular patterns (PAMPs) by Toll-like receptors (TLRs) plays a crucial role on multiple cell types to initiate protective immune responses. This study is aimed at delineating the direct effects of each TLR signaling on B cells.
Multiple toll-like receptors (TLR1, 2, 3, 4, 6, 7 and 9) are expressed on B cells. We isolated mature, naïve B cells from mouse spleens and stimulated them in vitro with individual TLR agonists alone or in combination with T cell help (via ligation of CD40) and TH2 cytokine interleukin IL-4. B cell responses including proliferation, antibody class switch recombination (CSR) and cytokine production were assayed. We confirmed that stimulation of TLR4 on B cells by lipopolysaccharide (LPS) potently induced robust proliferation and CSR to IgG3. Stimulation of TLR1/2 and 2/6 by agonists Pam3CSK4 and FSL-1 respectively, induced proliferation and CSR to IgG3 albeit to a lesser degree compared to LPS. Stimulation of TLR9 by CpG oligodeoxynucleotides (ODNs) induced robust proliferation, but did not promote CSR. In the presence of anti-CD40 and IL-4, stimulation of TLR1/2, TLR2/6, or TLR9 enhanced proliferation and CSR to IgG1. In contrast, TLR3 agonist (poly I:C) and TLR7 agonist (single-stranded RNA) had no detectable effects on proliferation or CSR. Investigations of TLR signaling on cytokine production by B cells are ongoing. Altogether, these results demonstrate that stimulation of TLRs differentially induce B cell proliferation and modulate CSR.
Physiological V(D)J Recombination is Mediated by RAG Scanning of Loop-extruded Chromatin
Authors: Zhang Yu , Zhang Xuefei , Liang Zhuoyi , Ba Zhaoqing , Dring Eddie , Zurita Jeffrey , Aiden Aviva Presser , Aiden Erez Lieberman , Alt Frederick W. 
Published: January 2023

Abstract
RAG endonuclease initiates V(D)J recombination by cleaving paired V, D, and J gene segments flanked by complementary recombination signal sequences (RSSs). We address physiological relevance of RAG scanning and the underlying mechanisms through studies of the ~300 kb 3′-proximal IgH locus CBE-anchored loop domain. This domain contains nine Ds and four JHs within a 57-kb region between the upstream IGCR1 loop anchor and the V(D)J recombination center (RC). IgH D to JH-rearrangements occur in deletional orientation, mediated by convergent downstream D-RSSs and JH-RSSs, despite Ds having upstream RSSs in the same orientation as JH-RSSs that could promote inversion. Extra-chromosomal V(D)J recombination substrate studies attributed this deletional orientation-specific joining bias to D-RSS sequence differences. However, we find that deletional joining of 8 of 9 chromosomal IgH Ds, results from RAG linear scanning of chromatin upstream of the RAG-bound RC, a process that detects convergent D-RSSs but to which D-RSSs in the same orientation are invisible. The exceptional D employs an RSS-based mechanism to counteract proximal location to JHs in the RC. By deleting JHs, we reveal that RAG scans directionally downstream from 3′D-RSSs until reaching the IgH 3′CBE loop anchor. Targeting catalytic-dead Cas9 (dCas9) binding in this scanning path impedes RAG scanning in association with cohesin accumulation and loop formation between the block site and RC. Strong transcription through a repetitive downstream switch region blocks RAG scanning similarly to the dCas9 block. We demonstrate a critical role for RAG scanning in physiological V(D)J recombination and implicate cohesin-mediated loop extrusion as a driving force.