bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and application of ChIPSeq: genome‐wide DNA binding by MyoD
Authors: Kwan Gordon , Sharp Sandra B , Wold Barbara J
Published: June 2021

ChIPSeq is an application of high throughput direct sequencing (Solexa / Illumina) to assay for factor‐DNA binding sites retrieved by chromatin immunoprecipitation (ChIP). A goal was to achieve reliable identification of true binding sites throughout large genomes, without need for cloning or microarray. The target protein MyoD, a muscle‐specific transcription factor, was chosen for proof‐of‐principle for the large class of factors that bind small and degenerate motifs (e.g. CANNTG) and to acquire new information regarding the role of MyoD in myogenesis. For ChIP optimization, MyoD‐ChIP DNA fragments were assayed by quantitative (real time) PCR using a set of locations known to be positive for MyoD binding. For the direct sequencing assay, MyoD‐ChIP and control normal mouse IgG‐ChIP fragments were cluster‐amplified and sequenced. 32‐nt sequence reads were mapped to the mouse genome. MyoD‐ChIP and control reads were analyzed jointly to identify enriched regions. 6.8M out of 17.4M total sequence reads mapped to single locations. 5,113 genes were identified from 13,194 enriched regions. Data revealed expected and unexpected MyoD binding site locations. The results suggest that the method is broadly applicable for identifying binding locations and thereby identifying binding sequence motifs of any transcription factor in any sequenced genome, large or small. CSUPERB Joint Venture Grant to SBS.
Leukocyte‐derived Myeloperoxidase modulates the expression of multiple Hepatic Gene Families during acute systemic Inflammation
Authors: Vasu Vihas T , Hemmad Aseem R , Kubala Lukas , Rathnayake Samira , Gohil Kishorchandra , Cross Carroll E , Eiserich Jason P
Published: June 2021

Myeloperoxidase (MPO) is a hemoprotein/enzyme abundantly expressed by leukocytes [neutrophils, macrophages, kupfer cells (liver)] that catalyzes the formation of free radical species implicated in inflammatory processes. The capacity of MPO to modulate gene expression during inflammatory events remains incompletely characterized. We have compared global hepatic gene expression profiles in male wild‐type (MPO+/+) and MPO‐deficient (MPO−/−) mice subjected to endotoxemia. Male mice were euthanized 24 hrs following i.p. injection of either sterile saline or E. coli LPS (10 mg/kg), liver tissue harvested, mRNA extracted and subjected to microarray analysis using Affymetrix 430 2.0 GeneChips. Global gene expression analysis demonstrated that the number of differentially expressed genes in LPS treated MPO−/− mice were dramatically higher (3156 genes) than MPO+/+ mice (1519 genes). The data showed a LPS‐induced upregulation of genes related to inflammatory‐immune responses in both MPO+/+ and MPO−/− mice. Genes related to metabolic processes were downregulated in both MPO+/+ and MPO−/− mice treated with LPS, where the effect was more in MPO−/− mice (342 genes) as compared to MPO+/+ mice (128 genes). The present data suggest that inflammatory and metabolic processes are co‐ordinately dysregulated in LPS‐challenged MPO−/− mice and provide documentation of novel genes which might be regulated by MPO.
Classes of calcium sensing receptor inactivating mutations define trafficking defects
Authors: Breitwieser Gerda E. , White Elissa
Published: June 2021

The calcium sensing receptor (CaR) is a Family C GPCR that translates changes in extracellular Ca2+ into diverse intracellular signals. Inactivating mutations in human CaR cause Ca2+ handling diseases including familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT). During biosynthesis, CaR must navigate a number of quality control checkpoints, including glycosylation and dimerization, or be shunted to the endoplasmic reticulum‐associated degradation pathway. We have shown that CaR must also navigate a conformational or functional checkpoint in the ER, i,e., activating mutations and/or an allosteric agonist protect against degradation and increase plasma membrane CaR while inactivating mutations and/or an allosteric antagonist increase ERAD. We have examined the biosynthesis of CaR mutations causing FHH/NSHPT by immunoprecipitation, biotinylation, and functional assays. We define several classes of CaR mutants based on rescue of total expression and plasma membrane localization upon treatment with the proteasomal inhibitor MG132. Most interesting are the CaR mutants which show both increased total expression and enhanced plasma membrane localization with MG132, permitting assay of functional activity. Some inactivating mutations of CaR cause a trafficking defect which can be corrected by either proteasome inhibition or treatment with allosteric agonist. Supported by NIH GM077563.
Effect of Apolipoprotein E glycosylation on lipoprotein and Aβ metabolism in an animal model for Niemann‐Pick Type C disease
Authors: Wong Boon‐Seng , Chua Ching‐Ching , Lim Mei‐Li , Ho Jaqueline Zyet‐Ee
Published: June 2021

Neurodegeneration is the final cause of death in Niemann‐Pick Type C (NPC) disease, a cholesterol‐storage disorder. Accumulating evidence indicates that NPC may share common pathological mechanisms with Alzheimer's disease (AD), including the link between cholesterol and amyloid‐ß (Aß) deposition. However, the mechanisms underlying neurodegeneration in NPC is unclear. Apolipoprotein E (apoE) is highly expressed in the brain and plays a pivotal role in cholesterol metabolism. Altered apoE expression is associated with defective interaction with lipoproteins and causes hypercholesterolemia, a risk factor for AD. Moreover, ApoE polymorphism was found to modulate the susceptibility to develop AD by differentially binding to Aß and regulating their clearance. Earlier studies showed that ApoE is differentially sialylated but the functional significance of this chemical alteration on ApoE role in cholesterol homeostasis and Aß catabolism is unknown. In this study using an animal model of NPC, we will correlate the glycan profile on neuronal ApoE with Aß accumulation and cholesterol dysfunction at different stages of disease development. We will also profile the changes in the biochemistry of lipoprotein metabolism and in the expression of enzymes involved in Aß metabolism during the neurodegenerative cascade.
TTC4 provides a functional link between RASSF1C and DNA primase
Authors: Dmitriev Ruslan , Shakhparonov Mikhail I , Pestov Nikolay B
Published: June 2021

TTC4 protein was identified as a putative tumor suppressor since its gene is often mutated in some cancers. Also, its drosophila homologue was shown to interact with DNA primase (p180). Recently, we observed that murine TTC4 interacts with hampin, a component of MYST1 histone acetyltransferase complex. This interaction may provide a link between DNA primase (p180) and other tumor supressor proteins, such as RASSF1C, in the complex interactome network. We observed that in cultured 3T3 fibroblasts and other mammalian cell lines TTC4 localizes during cell cycle progression similarly to DNA primase (p180). In addition, the nuclear pool of endogenous TTC4 has a punctate pattern partly co‐localizing with endogenous RASSF1C and PML nuclear bodies.

Upon overexpression, TTC4 has almost exclusive cytoplasmic localization and its transport to nucleus occurs only in the presence of overexpressed RASSF1C. Interestingly, overexpressed TTC4 with RASSF1C also co‐localize on microtubules. Therefore, RASSF1C appears to be required for nuclear sorting of TTC4. We propose that this regulation of TTC4 nuclear sorting by RASSF1C may be important for its association with DNA primase (p180) and, therefore, may constitute a part of unknown signaling pathway leading to regulation of DNA synthesis.
Evaluating Multiple Signaling Activities in LPS Induced Human PBMCs
Authors: Whiteman Sarah Ann , Stapleton Suzanne , Jett Marti , Medis Chanaka
Published: June 2021

Lipopolysaccharide (LPS) is an endotoxin that induces the secretion of pro‐inflammatory cytokines in many cells which can induce septic shock in humans. This project is aimed at developing a rapid diagnostic method to combat the effects of LPS by utilizing signal transduction pathways induced by LPS in Peripheral Blood Mononuclear Cells (PBMC) as a prototype module. A set of LPS induced genes were identified through micro array analysis and common signaling pathways were found. Further investigation of the pathways led to the identification of common components to multiple pathways that can serve as diagnostic markers. We analyzed two targets by first evaluating the time dependent gene expression pattern and then by investigating the impact on components that are indirectly associated with the targets. We will further evaluate the specific inhibitors by correlating the observed gene expression pattern to the protein expression using ELISA. We believe the study will allow us to better understand the complex interactions of multiple signal transduction pathways induced by LPS.
Prostaglandin E2 inhibits lung fibroblast proliferation and collagen expression via distinct cAMP effector pathways: differential roles of PKA and Epac‐1
Authors: Huang Steven Kenneth , Wettlaufer Scott H. , Chung Jooho , Peters‐Golden Marc
Published: June 2021

Fibroblasts are key effectors in the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis. Prostaglandin (PG) E2 inhibits lung fibroblast collagen production and proliferation through receptor ligation and production of the second messenger cAMP, but the downstream pathways mediating such actions have not been fully characterized. We sought to investigate the roles of the cAMP effectors protein kinase A (PKA) and exchange protein activated by cAMP‐1 (Epac‐1) in modulating these functions in primary human fetal lung IMR‐90 fibroblasts. Treatment with a cAMP analog that selectively activates PKA downregulated collagen I production but not proliferation, while an Epac‐selective analog downregulated proliferation but not collagen. These selective roles of PKA and Epac‐1 were confirmed by treatment with the PKA inhibitor KT 5720, overexpression of the PKA catalytic subunit, and silencing Epac‐1 using short hairpin RNA. The small GTPase Rap1 was activated by PGE2 and the Epac agonist but not the PKA agonist; decreasing Rap1 activity by transfecting with Rap1GAP blocked the ability of PGE2 to inhibit proliferation. We conclude that PKA and Epac‐1 differentially mediate the ability of PGE2/cAMP to inhibit distinct aspects of fibroblast activation.
Molecular Approaches to Promote Wound Healing in Sulfur Mustard Exposed Human Keratinocytes In Vitro: Role of Nitric Oxide in Wound Healing
Authors: Ishida Hiroshi , Ray Radharaman , Ray Prabhati
Published: June 2021

Sulfur mustard (SM), a chemical warfare agent, produces burns with sever skin blistering. Promoting re‐epithelialization is a challenging issue in the SM skin injury treatment. We reported that human inducible nitric oxide (NO) synthase (iNOS) was a factor for skin wound healing after SM exposure in the normal human epidermal keratinocytes (NHEKs) scratching‐wound model (Ishida et al., J Dermatol Sci, 2007). In this study, we carried out experiments to further elucidate the roles of iNOS/NO by manipulating iNOS gene expression with siRNA and adenovirus constructs for gene knockdown and overexpression, respectively. NOC‐12, a NO donor drug and L‐NIL, an iNOS inhibitor, were also tested to study the roles of iNOS. The siRNA transfection significantly reduced both the gene and protein expression levels followed by a complete inhibition of wound healing. Infection of NHEKs with the adenovirus constructs resulted in a marked increase of iNOS expression in a time‐ and dose‐dependent manner. NOC‐12 reversed wound healing suppressed by the siRNA or L‐NIL; however, neither overexpression nor NO donor application caused significant improvement of wound healing inhibited by SM.

These statistics suggest that SM causes not only the pathway involved NO production, but also another pathway independent from NO production.

This work was supported by JSTO‐CBD/DTRA award # 2.T0002_05_WR_C to Prabhati Ray.
Nitric oxide (NO) signaling modulates intracellular calcium [Ca2+]i and prevents alcohol‐induced neuronal death in cerebellar granule cell (CGN) cultures
Authors: Kouzoukas Dimitrios Elias , Li Guiying , Takapoo Maysam , Bhalla Ramesh , Pantazis Nicholas J.
Published: June 2021

One of alcohol's most damaging effects on the developing CNS is severe neuronal loss. For unknown reasons, the cerebellum and hippocampus are more vulnerable to alcohol‐induced neuronal loss than other regions. One possibility is that alcohol‐resistant regions have protective mechanism(s), which mitigate alcohol toxicity. Our prior studies revealed that NO is protective, reducing alcohol‐induced neuronal loss by activating a NO‐signaling pathway (NO activates guanylyl cyclase; the increase in cGMP activates cGMP‐dependent protein kinase, PKG). A current goal is to identify a downstream target for PKG. We are exploring interactions among PKG, alcohol‐induced neuronal death, and [Ca2+]¡ for several reasons. Alcohol disrupts [Ca2+]¡ homeostasis, and neurons are sensitive to calcium disruption. PKG regulates the inositol 1,4,5‐triphosphate receptor (IP3R), modulating [Ca2+]¡ release. Ethanol (400 mg/dl) has two effects on CGN cultures: 1) A 25% reduction in neuronal numbers; 2) A rapid (< one min) increase in [Ca2+]¡. Calcium chelators (BAPTA‐AM; EGTA‐AM) decrease alcohol‐induced neuronal loss (<10%). An inhibitor of the IP3R (2‐APB) and a PKG activator (8‐Br‐cGMP) reduce both the alcohol‐induced [Ca2+]¡ surge and neuronal death (<5%). In summary, modulating [Ca2+]¡ reduces alcohol toxicity. NO may protect neurons by preventing alcohol's disruption of [Ca2+]¡. Support: NIAAA grant AA011577
Phosphorylation of the autoinhibitory insert in endothelial nitric oxide synthase promotes calmodulin binding at low free Ca2+ concentrations
Authors: Tran Quang‐Kim , Leonard Jared , Black DJ , Persechini Anthony
Published: June 2021

The 45‐residue autoinhibitory insert in the endothelial nitric oxide synthase (eNOS) reductase domain inhibits electron transfer, and this effect is counteracted when CaM is bound to the enzyme. We have investigated the effects of phosphorylation at Ser‐617 and Ser‐635 in this region on the Ca2+ sensitivities of CaM binding and CaM‐dependent enzyme activation and on reductase and synthase activities. The mean EC50(Ca2+) values (± SD) for CaM binding to bovine eNOS and mutants with S617D, S635D, S617D/S635D substitutions or lacking the entire autoinhibitory insert were determined to be 220 ± 25, 109 ± 7, 202 ± 21, 117 ± 14, or 47 ± 9 nM, respectively. Corresponding mean EC50(Ca2+) values for eNOS activation were found to be 440 ± 55, 240 ± 28, 461 ± 56, 263 ± 28, or 124 ± 16 nM. The S617D substitution and deletion of the insert were found to increase CaM‐dependent synthase and cytochrome c reductase activities 2‐fold, and to respectively increase CaM‐independent reductase activity 3‐ and 6‐fold. The S635D substitution was found to have no significant effect on any property examined. These results indicate that phosphorylation at Ser‐617 acts synergistically with CaM to counteract the effects of the autoinhibitory insert. Given a resting free Ca2+ concentration in endothelial cells of 50 – 150 nM, phosphorylation at this site also appears to promote binding of CaM to the synthase under basal conditions.

Source of research support: NIH Grant GM074887.
Hsp90 localized with Sp1 in mitosis involves in Sp1 stability
Authors: Wang Shao‐an , Hung Jan‐Jong
Published: June 2021

Heat Shock Protein 90 (Hsp90) localized within cytoplasm is an important chaperone to maintain its client proteins in a proper folding state. Previous studies revealed that most of its client proteins localized within cytoplasm. However, our previous study revealed that the interaction of Hsp90 and Sp1 localized major in nucleus is important for the expression of 12(S)‐lipoxygenase in A431 cells. How the Hsp90 co‐localized and affected the nuclear proteins is not clear. In the present study, we found that Sp1 and Hsp90 co‐localized during the mitotic period. Moreover, inhibition of Hsp90 by geldanamycin (GA) or Hsp90‐shRNA resulted in Sp1 degradation via an ubiquitination‐dependent pathway and Sp1 instability in mitosis. Our previous study has known that JNK1‐induced Sp1 phosphorylation at Thr278 and Thr739. In this study, we found that Hsp90 could facilitate the phosphorylation of Sp1 by JNK1, and then increase Sp1 stability in mitosos.
Screening for novel small molecule‐encoding sequences using environmental DNA cosmid libraries created from Pingry School soil metagenomes
Authors: DiMassimo Jack , Kim Jeff , Hata Tommie Shintaro , Brady Sean
Published: June 2021

The genomes of culturable bacteria have been a rich source of pharmaceutically useful genetically encoded small molecules. However, only a very small portion of bacteria are culturable using traditional laboratory methods (less than 1%) and recent shotgun‐sequencing projects has shown that the remaining species are a rich source of biosynthetic pathways that produce novel small molecules. To avoid culture‐dependent screening methods, environmental DNA cosmid libraries were constructed from soil metagenomes. Genes involved in a pathway producing a small molecule are often located adjacent to each other in the genome and cosmids can be used to isolate all of these genes into individual clones. The cosmid libraries were expressed in E. coli and screened in a high throughput fashion for clones producing genetically encoded small molecules. Since the small molecules often cause the bacteria to change in appearance, the clones can simply be screened for changes in appearance. In addition, overlay screens were utilized to identify clones producing small molecules capable of lysing other bacteria. This represents a rapid method to screen soil metagenomes for novel genetically encoded small molecules. This work was made possible by the Rockefeller University Summer Outreach Program and the Camille and Henry Dreyfus Foundation in the Chemical Sciences.
Cbfb‐SMMHC impairs differentiation of Common Lymphoid Progenitors and reveals an essential role for RUNX in early B cell development
Authors: Gerstein Rachel , Kuo Ya‐Huei , Castilla Lucio
Published: June 2021

The core‐binding factor (CBF)‐associated leukemia fusion protein CBFβ‐SMMHC impairs myeloid and lymphoid differentiation. By inhibiting RUNX function, the fusion oncoprotein predisposes specifically to acute myeloid leukemia in both patients and mouse models. We have shown that Cbfβ‐SMMHC expression leads to a sustained reduction of circulating B lymphocytes in the mouse. In this study, we demonstrate that the activation of Cbfβ‐SMMHC reduces both pre‐pro‐B and pro‐ B cells approximately 3 fold, pre‐B cells over 10 fold and that this differentiation block is cell‐autonomous. The reduction of pre‐pro‐B cells coincided with an increase in apoptosis in this population. The number of common lymphoid progenitors (CLPs) were not affected, however, the expression of critical early B cell factors Ebf1, Tcfe2a, and Pax5 in CLPs were significantly reduced. In addition, Cbfβ‐SMMHC reduced Rag1 and Rag2 expression, and impaired V(D)J recombination in the CLPs. Furthermore, CLPs expressing Cbfβ‐SMMHC also show inhibition of B cell‐specific genes Cd79a, Igll1, VpreB1 and Blk. These results demonstrate that CBF/RUNX function is essential for the function of CLPs, the survival and differentiation of pre‐pro‐B cells, and the establishment of a B lineage‐specific transcriptional program.
EBF restricts alternate lineage options and promotes B cell fate commitment independently of Pax5
Authors: Lancki David William , Pongubala Jagan M.R. , Northrup Daniel L. , Medina Kay L. , Treiber Thomas , Bertolino Eric , Thomas Matthew , Grosschedl Rudolf , Allman David , Singh Harinder
Published: June 2021

Alternate lineage restriction and B cell fate commitment require the transcription factor Pax5, but the functions of EBF, in these processes remain largely unexplored. In the absence of EBF, expandable and clonal lymphoid progenitors retain considerable myeloid potential. Conversely, ectopic expression of EBF in multipotential progenitors directs B cell generation at the expense of myeloid cell fates. EBF induces Pax5 and antagonizes expression of C/EBPα, PU.1 and Id2. Importantly, sustained expression of EBF in Pax5−/− hematopoietic progenitors is sufficient to block their myeloid and T lineage potentials in vivo. Furthermore, in Pax5−/− pro‐B cells increased EBF expression represses alternate lineage genes. Thus, EBF can restrict alternate lineage choice and promote B cell fate commitment independently of Pax5.
Variegation of CD4+ T cell phenotypes is regulated by IL‐12 and IFNα
Authors: Davis Ann M , Ramos Hilario , Matthews Loderick A. , Farrar J. David
Published: June 2021

Innate cytokines secreted by antigen presenting cells shape adaptive immune responses to pathogens. For example, viral infections promote robust production of both IL‐12 and IFNα/β, two cytokines which can regulate CD4+ T helper (Th) cell differentiation and effector function. We have demonstrated that IL‐12 is exclusively responsible for directing Th1 development and effector functions in human CD4+ T cells. In contrast to previous reports, IFNα alone was insufficient to promote Th1 development due to inability to maintain stable T‐bet expression. Furthermore, IFNα neither enhanced nor repressed IL‐12‐induced Th1 development. However, IFNα greatly enhanced IL‐12‐induced IL‐2 secretion in human Th cells. Furthermore, while IL‐12 promoted elevated expression of CXCR3, IFNα enhanced expression of CCR7. In cells activated in the presence of both IL‐12 and IFNα, a phenotypic variegation was observed in which IL‐2‐producing cells possessed a CXCR3loCCR7hi phenotype, while IFNγ‐producing cells were CXCR3hiCCR7lo. These phenotypic characteristics correlated with function, such that cells sorted for IL‐2 expression possessed greater capacity and phenotypic plasticity than cells sorted for IFNγ xpression. These results suggest a novel role for IFNα/β in the differentiation of Th cells to effector and memory phenotypes. Supported by grants NIH/NIAID AI056222 and NIH/GM GM00820317.
IL‐6 reveals an intrinsic polyspecific phenotype of the HEL T cell repertoire in BALB/c mice
Authors: Gabaglia Claudia Raja , Menezes Juscilene , Braciak Todd , Sercarz Eli
Published: June 2021

The T cell repertoire raised by immunization with hen egg lysozyme (HEL) in H‐2d mice has been well characterized by proliferation studies, as presenting a single dominant determinant (peptide108–116) and 2 subdominant determinants (p11–25 and p20–35). T cells directed to other areas on the molecule do not arise after HEL‐CFA immunization as detected in a pepscan by proliferation and, in our hands, by ELISAspot assays.

In previous experiments, IL‐6 had been shown to alter the hierarchy of peptide responses to HEL (Drakesmith et al, PNAS, 2000). We therefore immunized BALB/c mice with a CFA emulsion containing HEL with recombinant IL‐6 (or HEL‐CFA co‐immunized with an adenovirus vector containing IL‐6) to ask which T cells would be primed that could be recalled in vitro by a panel of overlapping 15 aa peptides all across the HEL molecule. IL‐6 plus HEL induced co‐dominant responses recalled by pepscan peptides in the regions 103–121, 67–85 as well as 87–103, as measured by proliferation and ELISAspot. Other, weakly subdominant determinants were also detected by proliferation and cytokine analysis in distinct areas: 9–25, 27–45, 37–51 and 47–61. Surprisingly, immunization with p61–85 primed for a response to p106–120 as well as to HEL. T cell hybrids generated after immunization with p106–120 and selected on the basis of reactivity to HEL, confirmed these results in that crossreactive responses to 106–120, 85–101 and 67–85 regions were displayed by public T cell clones. This phenomenon may be more frequent than previously imagined, and in this case extended to three determinants on the same molecule. Such polyspecific clones may achieved dominance owing to their polyreactive phenotypes. In conclusion, the inherent degenerate recognition of particular T cells can be revealed during inflammatory processes upregulated by agents such as IL‐6.

Grant support NIH R0142396
Characterization of B cell subsets in cynomolgus monkeys
Authors: Van Alstine Bishop Caroline Donna , Lin Zhonghua , Rangell Linda , Fuh Franklin , Danilenko Dimitry , Berry Karen , Balazs Mercedesz , Williams Marna
Published: June 2021

Plasma cells are believed to play a prominent role in the pathogenesis of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. In support of biomarker identification for B‐cell‐targeted therapies, we used an intracellular FACS‐based assay for the endoplasmic reticulum (ER)‐associated plasma cell marker PC (VS38c) and intracellular immunoglobulin to characterize plasma cells in non‐human primates. We identified two distinct subsets of PC‐expressing cells in cynomolgus monkey spleen – a population that expresses high levels of PC and IgG or IgM (PChighIghigh), and another subset that expresses intermediate levels of PC and IgG (PCintIgGint). Similar to human CD138+ plasma cells, the PChigh and PCint populations do not express CD20, express low levels of the BAFF receptor‐3 (BAFF‐R, BR3), and appear to be larger and more granular than PCneg B‐cells. There are increased percentages of Ki67+ proliferating cells in the PChighIgGhigh subset compared to the PCintIgGint population. The characterization of plasma cell populations in non‐human primates will expand our knowledge of B‐cell development and contribute to the development of novel therapies for autoimmune and inflammatory diseases.
Costimulatory Requirements for the Expansion of Human T Regulatory Cells
Authors: Golovina Tatiana Nikolaevna , Mikheeva Tatiana , Shan Xiaochuan , Suhoski Megan , Liu Ronghua , Case Chanelle , June Carl , Riley James
Published: June 2021

We used cell‐based artificial APCs (aAPC) to explore the costimulatory requirements to expand functional T regulatory cells (Treg) enriched from human peripheral blood. Only CD28 costimulation maintained high levels of FoxP3 expression and suppressive function in enriched T regulatory cultures. Interestingly, CD4 T cells grown in the presence of CD28 costimulation and rapamycin displayed significant in vitro suppressive activity. However, when these cells were evaluated for suppressive activity in vivo, only the cultures that were initially enriched for Tregs suppressed. We investigated whether additional costimulatory pathways could augment CD28's ability to promote further Treg expansion in rapamycin. In a minority of donors OX40 costimulation was able to augment Treg expansion and maintain function; however, in the majority of donors these cultures often lost suppressive function. In contrast, we found that re‐stimulation of the Treg cultures after 8–10 days of culture with CD28 costimulation alone resulted in 1000 fold expansion of Treg in less than 3 weeks. These highly expanded Tregs retained potent in vitro and in vivo suppressive function. These studies indicate the optimal costimulatory requirements to expand Tregs differ from T effector cells and establish the feasibility of using expanded Tregs for adoptive T cell therapy.

The project is supported by JDRF for Cell Therapy grant.
An efficient new column‐free immunomagnetic isolation method for mouse CD4+CD25+ regulatory T cells
Authors: Guilbault Benoit G. , Yuan Ning , Fairhurst Maureen A. , Eaves Allen C. , Thomas Terry E.
Published: June 2021

Mouse CD4+CD25+ regulatory T cells (Tregs) are extremely difficult to isolate due to their rareness and the absence of a unique marker that differentiates them from other cell types. Their isolation typically requires at least two steps, often including sorting by FACS, which can be time consuming and very expensive. We thus sought to develop a simple and efficient method for Treg isolation using column‐free immunomagnetic cell separation (EasySep®). The resulting two‐step method begins with depletion of non‐CD4+ T cells, and is followed by selection on CD25 whereby both CD4+CD25+ and CD4+CD25neg T cell fractions can be isolated to a high level of purity (90.3 ± 3.4% CD4+CD25+, n=14). This new method proved to be highly efficient as cell output was routinely well over 10e06 Tregs per spleen. Intracellular flow cytometric analysis of FOXP3 expression clearly demonstrated that isolated CD4+CD25+ T cell fractions were highly enriched for FOXP3 expressing cells (n=4). In vitro culture assays also showed that isolated CD4+CD25+ T cells were able to strongly suppress CD4+CD25neg T cell proliferation responses (n=3). We are currently working towards fully automating the CD25 positive selection step using the RoboSep® cell separator. This will add further convenience to a method that enables the efficient isolation of highly purified CD4+CD25+FOXP3+ Tregs from mouse lymphoid tissues.
HOZOT, a novel type of human regulatory T cell lines, showed opposing activities, helper and suppressor, depending on the stimulations of dendritic cells or anti‐CD3 antibody
Authors: Sugimoto Akira , Suzuki Motoyuki , Tsuji‐Takayama Kazue , Yamamoto Mayuko , Harashima Akira , Inoue Toshiya , Otani Takeshi , Takeuchi Makoto , Nakamura Shuji , Kibata Masayoshi
Published: June 2021

HOZOT cell lines are one type of the regulatory T cells (Treg) established from human umbilical cord blood (UCB) without cytokines. These HOZOT cells have multi‐functional activities, not only suppressor but also cytotoxic activity. In this study, we have examined another novel function of HOZOT in immune system. Cord blood naive CD4+ T cells as responder cells were stimulated with dendritic cells (DC) or plate coated anti‐CD3 antibody (anti‐CD3 Ab) in the presence of HOZOT‐4. The DC‐induced proliferation of naive CD4+ T cells derived from UCB was inhibited by the addition of HOZOT‐4. Whereas, anti‐CD3 Ab‐induced proliferation was promoted by the same treatment. Anti‐CD3 Ab ‐cultures have produced IFN‐γ, not IL‐4. Opposing effects of HOZOT‐4 were unique because naturally occurring Treg inhibited both DC‐ and anti‐CD3 Ab‐induced stimulations. Kinetics study showed that cell division was first detected at day 3 and increased later on. IL‐2 was not involved in the mechanism of helper activity of HOZOT‐4 because no IL‐2 was detected in the anti‐CD3 Ab‐induced coculture system and blocking antibodies for IL‐2 and IL‐2R did not affect helper activity. Moreover, this helper activity could not be inhibited with blocking reagents for costimulatory molecule of CD28/B7. This helper activity exerted by HOZOT may be mediated through a novel costimulatory pathway.
A role for MMP‐9 in CD8+ T regulatory suppressor function
Authors: Taheri Faramarz , Stein‐Streilein Joan
Published: June 2021

Matrix metalloproteinase 9 (MMP‐9) belongs to a family of enzymes known for their ability to degrade extracellular matrix components. Macrophages, neutrophils and epithelial cells express MMP‐9 and use MMP‐9 to travel through tissues. However, MMP‐9 has other biological functions such as activating TGFβ. A recent report from our laboratory showed that CD8+ T regulatory cells upregulated MMP‐9 mRNA during the induction of peripheral tolerance through the eye. To test the postulate that MMP‐9 has a role in CD8+ Treg activity, we examined MMP‐9 KO mice and determined that they were unable to develop peripheral tolerance to anterior chamber inoculated antigen. Because MMP‐9 could be involved in trafficking to the site of antigen as well as suppression of T cell effector function, we adoptively transferred in vitro generated Treg cells directly into the ears of mice challenged with immune cells and antigen. Unlike the CD8+ Treg cells from wild type mice, the Treg cells generated with MMP‐9 KO were unable to suppress Th1 responses (delayed hypersensitivity). Our findings indicate that CD8+ Treg cells must express MMP‐9 for optimal suppressor activity. Since MMP‐9 also has the ability to activate TGF, the possibility is raised that CD8+ Treg derived MMP‐9 contributes to the activation of latent TGFβ in the CD8+ Treg cell which in turn contributes to the suppression of the Th1 response. Supported by NIH: EY 016476.
CD4+FoxP3+ regulatory T cells confer infectious tolerance in a TGF‐β dependent manner
Authors: Andersson John , Tran Dat , Shevach Ethan M
Published: June 2021

CD4+FoxP3+ regulatory T cells (TR) comprise a separate lineage of T cells that are essential for maintaining immunological tolerance to self. One molecule that has been extensively studied in TR suppression is TGF‐β, but its importance remains controversial. To further analyze the contribution of TGF‐β in TR function, we have used recombinant TGF‐β latency associated peptide (LAP) to neutralize active TGF‐β. LAP modestly reversed the non‐responsive state of CD4+CD25+ T cells from conventional mice, but had no effect on Foxp3+ TR from Foxp3‐GFP knock in mice, suggesting that TR associated TGF‐β plays a role in suppressing the responses of activated T cells. Co‐culture of pre‐activated Foxp3+ TR and naïve Foxp3−CD4+ T cells in the presence of anti‐CD3 and IL‐2 results in the induction of Foxp3 expression in 10–20% of the responder cells. This conversion is dependent on TGF‐β as it can be blocked with LAP and requires contact between the TR and effector T cell. The induced CD4+FoxP3+ T cells are suppressive when co‐cultured with fresh CD4+Foxp3− T cells in vitro and when mixed with CD4+Foxp3− T cells in vivo. Thus, it appears that TR associated TGF‐β can play a role in suppression of activated effectors, but more importantly can mediate infectious tolerance by converting conventional T cells into TR.
Anergy in Septic Patients: Correlating the Increased Percentage of Circulating CD4+CD25+CD127‐Regulatory T Cells with a Decline in Lymphocyte Proliferation
Authors: Venet Fabienne , Chung Chun‐Shiang , Lepape Alain , Ayala Alfred , Monneret Guillaume
Published: June 2021

Septic shock is the leading cause of death in intensive care units in the developed world. Septic patients initially up regulate both pro‐inflammatory and counter‐regulatory responses, the latter predominating long after the onset of sepsis. Previously we reported that while CD4+CD25– cells decline, the number of circulating CD4+CD25+ regulatory T cells (Treg) persists in these patients. However while the potent anti‐inflammatory properties of these patients’ Tregs has been documented, their actual involvement in sepsis‐induced immune dysfunction remains unclear. Here using flow cytometry we attempted to determine the contribution of Treg to immune dysfunctions observed after septic shock. Using whole blood samples we observed that Treg in healthy individuals and patients expressed Foxp3 and exhibited regulatory properties. Importantly, their percentage increased in patients and was negatively correlated with decreased lymphoproliferative response. Further using a polymicrobial septic mouse model, we also found an increased percentage of Treg and decline in splenocyte‐proliferation, which was ameliorated by ex vivo Foxp3 siRNA transfection. Taken together, these data suggest not only that Treg may play an active role in suppressing lymphoproliferative responsiveness after septic shock, but also that their rise in frequency may be a useful whole blood diagnostic marker of septic patient anergy.
Role of the 3’ heavy chain enhancers in germline transcription and switch recombination
Authors: Dunnick Wesley A , Shi Jian , Collins John T
Published: June 2021

To study germline transcription and DNA recombination associated with class switch recombination, we utilize transgenic mice with a 230 kb BAC that includes a rearranged VDJ gene, the entire heavy chain constant region locus, and all known 3 enhancer elements. We have identified a transgenic line that retains the HS3a 3. enhancer, but lacks HS1,2, HS3b, HS4, and other sequences more 3. This transgene expresses transgenic μ transcripts and IgM protein, but fails to produce germline transcripts or undergo switch recombination to the γ3, γ2b, γ2a, or ε genes. The heavy chain transgene with only HS3a expresses germline transcripts of both the γ1 and α genes, and undergoes switch recombination to α, but not to γ1. We have also derived three different sets of transgenic mice with engineered versions of the BAC heavy chain locus: (i) with loxP sites 5. of HS3a and 3. of HS4, allowing deletion of 28 kb including HS3a to HS4, (ii) one kb deletions of HS1,2 and HS3b, and HS4 flanked by loxP sites, and (iii) one kb deletions of HS3a, HS1,2, and HS3b, and HS4 flanked by loxP sites. We will report on heavy chain gene expression from these transgenes, both before and after Cre‐mediated deletion.
Regulation of the polymeric immunoglobulin receptor by different phyla of colonic bacteria
Authors: Rogier Eric W. , Bruno Maria E.C. , Kaetzel Charlotte S
Published: June 2021

The lower gastrointestinal tracts of mammals are exposed daily to hundreds of trillions of beneficial bacteria. Secretory IgA antibodies are transported into the intestinal lumen by the polymeric immunoglobulin receptor (pIgR), where they influence the composition of the intestinal microbiota, prevent bacterial invasion, and contribute to mucosal homeostasis. To compare the stimulatory effects of the 4 major bacterial phyla found in the human gut, mRNA levels for pIgR, the pro‐inflammatory factors TNF and IL‐8, and A20, a negative regulator of NF‐κB, were measured in the human colonic epithelial cell‐line HT‐29 following co‐culture with heat‐killed bacteria. Lactobacillus acidophilus (a Gram+ Firmicute) and Bacteroidetes thetaiotamicron (a Gram‐ Bacteriodete), representing the two most abundant phyla of gut bacteria, did not stimulate gene expression. By contrast, Escherichia coli strain Nissle (a Gram‐ Proteobacterium) and Bifidobacterium longum (a Gram+ Actinobacterium), representing less abundant phyla, dramatically induced gene expression. Levels of TNF, IL‐8 and A20 mRNA peaked at 3 h and were down‐regulated by 24 h, whereas pIgR expression peaked at 24 h, indicating the use of different regulatory pathways. These results suggest that the composition of the colonic microbiota may alter the expression of pro‐ and anti‐inflammatory genes and influence mucosal homeostasis. Supported by the CCFA.
Formation of in vivo immunological synapses (IS) between CTLs and virally infected astrocytes leads astrocyte to polarization mediated by the activation of a Rho‐GTPase signaling pathway.
Authors: Lowenstein Pedro R , Puntel Mariana , Barrett Robert , Yang Jieping , Sanderson Nicholas , Bondale Niyati , Assi Hikmat , Kroeger Kurt M , Castro Maria G
Published: June 2021

Immunological synapses (IS) mediate intercellular communication between T cells and APCs. T cell polarization at the Kupfer‐type IS consists of a peripheral supramolecular activation cluster (pSMAC), which is high in LFA‐1, and a central SMAC, high in TCR. Target virally infected astrocytes also polarize towards the immunological synapse in vivo. Astrocyte polarization in response to physical lesions in vitro is mediated through the activation of a signaling pathway mediated by a family of Rho‐GTPases, and coordinated by Cdc42. Here we tested the hypothesis that that stimulation of a Rho‐GTPase cascade underlies astrocyte polarization in response to an immune attack in vitro and in vivo. To do so, we infected brain astrocytes with a novel adenoviral (Ad) vector expressing a dominant negative variant of Cdc42 (Cdc42DN) and Thymidine Kinase (TK), a marker of infected cells, and analyzed their responses to a T cell attack. We show that during an anti‐viral immune response, infected astrocytes contacted by CTLs become polarized through the activation of a Rho‐GTPase signaling pathway.

This work was funded by grants from NINDS/NIH.
Vα14i NK T Cells Participate to the Early Response to MCMV Infection.
Authors: Brossay Laurent , Wesley Johnna D. , Tessmer Marlowe S. , Chaukos Deanna
Published: June 2021

Immunity to the well‐characterized virus MCMV is critically dependent on the innate response for initial control of viral replication. The contribution of innate immune cells, other than natural killer (NK) cells, to the propagation of an optimal anti‐viral immune response has not been fully examined. Therefore, we sought to determine the role and function of a unique, innate subset of T cells, Vα14 invariant natural killer T (Vα14i NK T) cells, in the anti‐viral response to MCMV infection in vivo. Vα14i NK T cells become activated and produce significant levels of IFN‐γ but not IL‐4 following MCMV infection, particularly in the liver. Adoptive transfers of Vα14i NK T cells in MCMV infected CD1d17−/− mice demonstrate that CD1d1 is not critical for their activation. Interestingly, both IFN‐α/β and IL‐12 are partially required for their activation status. Vα14i NK T cell derived IFN‐γ is highly dependent on IL‐12 but surprisingly partially dependent of IFN‐α. Collectively, these findings highlight the unique activation requirements of these innate T cells in the induction of an anti‐viral immunity.

Supported by NIH grant AI46709.
Roles of CD1d‐restricted Vα 14+ NKT cells in Theiler's virus infection, a viral model for multiple sclerosis
Authors: Tsunoda Ikuo , Tanaka Tomoko , Taniguchi Masaru , Hasanovic Faris , Fujinami Robert
Published: June 2021

Most murine natural killer (NK) T cells express an invariant Vα 14 T cell receptor and are CD1d‐restricted. We investigated the contribution of NKT cells in an animal model for multiple sclerosis (MS), Theiler's murine encephalomyelitis virus (TMEV) infection. First, we treated susceptible SJL/J mice with an antibody against Vα 14 during TMEV infection. Treatment during the early stage of infection delayed the onset of clinical disease with relatively higher interleukin (IL)‐4 production. In contrast, treatment during the late stage or weekly injections resulted in more severe demyelination with higher virus persistence. Next, we compared TMEV infection in wild‐type versus CD1d KO mice in the background of TMEV‐resistant BALB/c mice. Although no wild‐type mice developed demyelination, 20 and 60% of CD1d KO mice developed demyelination at 3 and 5 weeks after infection, respectively. TMEV‐infected CD1d KO mice showed higher IL‐4 production and TMEV‐specific lymphoproliferative responses, compared with wild‐type mice. These results suggest that NKT cells could modulate inflammatory demyelinating disease by altering the cytokine profile and virus‐specific immune responses. This work was supported by the University of Utah (Funding Incentive Seed Grant to IT) and the National Institute of Health (NS34497 to RSF).
CD70 antibody therapy for the prevention and treatment of experimental Inflammatory Bowel Disease (IBD).
Authors: Manocha Monika , Rietdijk Svend , Laouar Amale , Bhan Atul , Terhost Cox , Swamy Manjunath N
Published: June 2021

Background

Inflammatory bowel disease is a chronic disorder of the gastrointestinal tract, characterized by an aberrant mucosal T cell‐mediated inflammation. The costimulatory molecule CD70 is constitutively expressed by a unique antigen presenting cell in the lamina propria and its interaction with T cells is critical for the gut mucosal T cell activation.

Objective

To test if blockade of CD70 reduces intestinal inflammation in an experimental model of colitis

Methods

Colitis was induced by adoptively transferring CD45RBhiCD4+ T cells into Rag 1−/− mice, the mice were treated with control Ig or a blocking anti‐CD70 antibody twice weekly for 6‐8 weeks and the animals were monitored for disease development.

Results

Treatment with CD70 blocking antibody dramatically reduced the colitis‐associated weight loss, clinical symptoms and intestinal hisopathology. Importantly, anti‐CD70 antibody treatment initiated after onset of the disease was also able to partially reverse the features of colitis. The Th‐1 related cytokine IFNγ as well as key proinflammatory cytokines of the Th17 pathway, including IL‐23, IL‐17, IL‐6, and TNFα were significantly reduced after CD70 antibody treatment as compared to control group (p<0.001). Conclusion: This study shows that both Th1 and Th17 related cytokines play a dominant role in the pathogenesis of IBD and that CD70 blockade can effectively suppress these cytokines leading to reduction of inflammation.

Source of Research support

Crohn's and Colitis foundation of America (CCFA)
Neutralizing epitope‐specific antibody responses in Anthrax Vaccine Absorbed (AVA) vaccinated individuals
Authors: Crowe Sherry R , Farris A Darise , Ash Linda L , Harley John B , James Judith A
Published: June 2021

The release of Bacillus anthracis spores can cause significant morbidity and mortality and an effective vaccine as well as directed immunotherapeutics are needed. The currently licensed vaccine requires six primary vaccinations, annual boosters, and has been associated with serious adverse events. Thus there is increasing interest in developing an improved vaccination strategy which can generate lasting protective immunity with reduced vaccinations. This study seeks to identify the major humoral targets of B. anthracis that provide protection. To this end, plasma was obtained from two hundred vaccinated individuals and thirty unvaccinated controls. Ninety‐five percent of all vaccinated individuals had anti‐protective antigen antibodies and the titer of these antibodies correlated with in vitro protection against lethal toxin. Utilizing overlapping decapeptides, we identified common antigenic regions within those individuals with high toxin neutralizing activity and demonstrated that these epitope‐specific antibodies were able to mediate protection in vitro. Thus we have identified an antigenic region within the protective antigen of Bacillus anthracis that provides protection in an in vitro lethal toxin assay. The findings from these humoral epitope‐binding studies have important implications for vaccine design and immunotherapeutic development. NIAID: 5U19AI062629
Melan‐A‐specific T cells derived from A2+ or A2‐umbilical cord blood (UCB) units proliferate and exhibit specific cytolytic function following in vitro stimulation
Authors: Merindol Natacha , Gimmig Sylvain , Duval Arnaud , Duval Michel , Champagne Martin A , Soudeyns Hugo
Published: June 2021

UCB is increasingly used as a source of haematopoietic progenitors to treat a variety of blood disorders requiring bone marrow transplantation. Advantages of UCB include availability and reduced HLA matching requirements as compared with bone marrow. To characterize functional properties of UCB T cells in the context of allogeneic UCB transplant, naïve CD8+ T cells specific for the HLA‐A2‐restricted Melan‐A‐derived peptide ELAGIGILTV were sorted from A2+ and A2− UCB units using A2/Melan‐A tetramers and were cultured in the presence of IL‐2, IL‐7, PHA, and peptide. Microcultures were phenotyped and tested for Melan‐A‐specific cytotoxicity by 51Cr‐release assay. Clonal diversity was estimated by T cell receptor (TCR) β chain sequencing. 83.3% (15/18) of clinical units tested were positively stained by tetramers (median frequency of 0.11% regardless of HLA type). Following in vitro stimulation, the median frequency of tetramer+ T cells was increased (p<0.0001), T cell microcultures exhibited Melan‐A‐specific cytolytic activity (median = 11.98% of positive cultures at an input of 1000 cells/well), and the TCR β chain repertoire showed evidence of oligoclonality consistent with antigen‐driven expansion. This study will bring new light on the ability of the preimmune T cell repertoire to participate in antitumoral and antiviral activity following UCB transplantation.

Supported by FRSQ/Héma‐Québec.