Publications by authors named "Charles Whitehurst"

18 Publications

  • Page 1 of 1

TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9.

Nature 2020 05 13;581(7808):316-322. Epub 2020 May 13.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2282-0DOI Listing
May 2020

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells.

Cell 2019 11 7;179(5):1144-1159.e15. Epub 2019 Nov 7.

Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO 63110, USA. Electronic address:

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904908PMC
November 2019

Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.

Cell 2019 09 29;178(6):1493-1508.e20. Epub 2019 Aug 29.

Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060942PMC
September 2019

Single Cell Explorer, collaboration-driven tools to leverage large-scale single cell RNA-seq data.

BMC Genomics 2019 Aug 27;20(1):676. Epub 2019 Aug 27.

Computational Biology, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT, 06877, USA.

Background: Single cell transcriptome sequencing has become an increasingly valuable technology for dissecting complex biology at a resolution impossible with bulk sequencing. However, the gap between the technical expertise required to effectively work with the resultant high dimensional data and the biological expertise required to interpret the results in their biological context remains incompletely addressed by the currently available tools.

Results: Single Cell Explorer is a Python-based web server application we developed to enable computational and experimental scientists to iteratively and collaboratively annotate cell expression phenotypes within a user-friendly and visually appealing platform. These annotations can be modified and shared by multiple users to allow easy collaboration between computational scientists and experimental biologists. Data processing and analytic workflows can be integrated into the system using Jupyter notebooks. The application enables powerful yet accessible features such as the identification of differential gene expression patterns for user-defined cell populations and convenient annotation of cell types using marker genes or differential gene expression patterns. Users are able to produce plots without needing Python or R coding skills. As such, by making single cell RNA-seq data sharing and querying more user-friendly, the software promotes deeper understanding and innovation by research teams applying single cell transcriptomic approaches.

Conclusions: Single cell explorer is a freely-available single cell transcriptomic analysis tool that enables computational and experimental biologists to collaboratively explore, annotate, and share results in a flexible software environment and a centralized database server that supports data portal functionality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-019-6053-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712711PMC
August 2019

Conformation constraint of anilides enabling the discovery of tricyclic lactams as potent MK2 non-ATP competitive inhibitors.

Bioorg Med Chem Lett 2013 Jun 4;23(11):3262-6. Epub 2013 Apr 4.

Medicinal Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.

Conformation restriction of linear N-alkylanilide MK2 inhibitors to their E-conformer was developed. This strategy enabled rapid advance in identifying a series of potent non-ATP competitive inhibitors that exhibited cell based activity in anti-TNFα assay.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2013.03.109DOI Listing
June 2013

Potency switch between CHK1 and MK2: discovery of imidazo[1,2-a]pyrazine- and imidazo[1,2-c]pyrimidine-based kinase inhibitors.

Bioorg Med Chem Lett 2013 May 4;23(10):2863-7. Epub 2013 Apr 4.

Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, USA.

Chemistry has been developed to access both imidazo[1,2-a]pyrazines and imidazo[1,2-c]pyrimidines. Small structural modifications in both series led to a switch of potency between two kinases involved in mediating cell cycle checkpoint control, CHK1 and MK2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2013.03.100DOI Listing
May 2013

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4.

Comb Chem High Throughput Screen 2012 Jul;15(6):473-85

Merck Research Laboratory, 33 Avenue Louis Pasteur, Boston, MA 02215, USA.

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/138620712800563945DOI Listing
July 2012

STAT6 phosphorylation inhibitors block eotaxin-3 secretion in bronchial epithelial cells.

Bioorg Med Chem 2012 Jan 17;20(2):750-8. Epub 2011 Dec 17.

Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge St., Boston, MA 02114, USA.

The STAT6 (signal transducer and activator of transcription 6) protein facilitates T-helper cell 2 (Th2) mediated responses that control IgE-mediated atopic diseases such as asthma. We have identified compounds that bind to STAT6 and inhibit STAT6 tyrosine phosphorylation induced by IL-4. In the bronchial epithelial cell line BEAS-2B, compound (R)-84 inhibits the secretion of eotaxin-3, a chemokine eliciting eosinophil infiltration. (R)-84 appears to prevent STAT6 from assuming the active dimer configuration by directly binding the protein and inhibiting tyrosine phosphorylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2011.12.006DOI Listing
January 2012

Discovery and Hit-to-Lead Optimization of Non-ATP Competitive MK2 (MAPKAPK2) Inhibitors.

ACS Med Chem Lett 2011 Aug 24;2(8):632-7. Epub 2011 Jun 24.

Merck Research Laboratories , 320 Bent Street, Cambridge, Massachusetts 02141, United States.

A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through high-throughput screening using the affinity selection-mass spectrometry-based Automated Ligand Identification System platform. Medicinal chemistry efforts optimized the initial screening hit to leadlike compounds with significant improvements in biochemical and cellular potencies, while maintaining excellent kinase selectivity and in vitro pharmacokinetic properties. Biophysical and biochemical studies confirmed the unique non-ATP-competitive binding mode of this series and suggested that highly selective inhibitors of MK2 should be feasible by targeting the outside ATP pocket.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ml200113yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017987PMC
August 2011

Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice.

BMB Rep 2008 Aug;41(8):575-80

Institute of Bioscience, Department of Bioscience and Biotechnology, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul 143-747, Korea.

Thymocyte-specific transcriptional regulatory systems can be used to better understand the relationship between transcription and V(D)J recombination during early T cell development. In this study, we generated transgenic mice expressing the transactivator Gal4-VP16 or the Gal4 DNA binding domain (Gal4-DBD) under the control of the lck proximal promoter, which is only active in immature thymocytes. From these studies Gal4-VP16 and Gal4-DBD expression was shown to significantly alter thymic cellularity and differentiation without significantly changing the CD3(+) thymocyte distribution. Furthermore, the presence of Gal4-VP16 or Gal4-DBD in the transgenic thymocytes retarded the mobility of the Gal4 DNA binding motif as determined by a gel mobility shift assay, suggesting that the developmental alteration did not affect the functional property of the transgenic proteins. These results indicated that lck promoter-driven Gal4-VP16 or Gal4-DBD expression did not affect CD3(+) mature thymocytes, thus this system can be applied to study transcriptional regulation of transresponder genes in bigenic mouse model thymocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5483/bmbrep.2008.41.8.575DOI Listing
August 2008

Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

Comb Chem High Throughput Screen 2008 Jul;11(6):427-38

Schering-Plough Research Institute, Cambridge, MA 02141, USA.

Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/138620708784911447DOI Listing
July 2008

Affinity selection-mass spectrometry screening techniques for small molecule drug discovery.

Curr Opin Chem Biol 2007 Oct 10;11(5):518-26. Epub 2007 Oct 10.

Schering-Plough Research Institute, 320 Bent Street, Cambridge, MA 02139, United States.

Affinity selection-mass spectrometry (AS-MS) techniques assess the binding of candidate molecules to immobilized or soluble receptors, and these methods are gaining acceptance in high throughput screening laboratories as valuable complements to traditional drug discovery technologies. A diversity of receptor types have been evaluated by AS-MS, including those that are difficult to screen using traditional biochemical approaches. AS-MS techniques that couple liquid chromatography-MS with size-based separation methods, such as ultrafiltration, gel permeation, or size-exclusion chromatography, are particularly amenable to the demands of MS-based screening and have demonstrated the greatest success across a broad range of drug targets. MS measurements of receptor function have many of the same advantages as AS-MS screening and are increasingly used for drug discovery as well.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpa.2007.07.011DOI Listing
October 2007

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry.

J Biomol Screen 2006 Mar 20;11(2):194-207. Epub 2006 Feb 20.

NeoGenesis Pharmaceuticals, Inc., Cambridge, MA 02139, USA.

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057105284340DOI Listing
March 2006

The T-cell receptor beta variable gene promoter is required for efficient V beta rearrangement but not allelic exclusion.

Mol Cell Biol 2004 Aug;24(16):7015-23

Center for Cancer Research and Department of Biology, Massachusetts Institute for Technology, Cambridge, MA 02139, USA.

To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the V beta 13 promoter was either deleted (P13(-/-)) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13(R/R)). In P13(-/-) mice, cleavage, rearrangement, and transcription of V beta 13, but not the flanking V beta gene segments, were significantly inhibited. In P13(R/R) mice, inhibition of V beta 13 rearrangement was less severe and was not associated with any apparent reduction in V beta 13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal V beta 13-recombination signal sequence junction and V beta 13 coding joint formation of both wild-type and mutant V beta 13 alleles. However, a low level of aberrant V beta 13 cleavage was consistently detected, especially in TCR transgenic P13(R/R) mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated V beta gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant V beta cleavages during allelic exclusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.24.16.7015-7023.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC479718PMC
August 2004

The T cell receptor beta enhancer promotes access and pairing of Dbeta and Jbeta gene segments during V(D)J recombination.

Proc Natl Acad Sci U S A 2003 Nov 30;100(23):13465-70. Epub 2003 Oct 30.

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The precise function of cis elements in regulating V(D)J recombination is still controversial. Here, we determined the effect of inactivation of the TCRbeta enhancer (Ebeta) on cleavage and rearrangement of Dbeta1, Dbeta2, Jbeta1, and Jbeta2 gene segments in CD4-CD8- [double-negative (DN)] and CD4+CD8+ [double-positive (DP)] thymocytes. In Ebeta-deficient mice, (i) Dbeta1 rearrangements were more severely impaired than Dbeta2 rearrangements; (ii) most of the Dbeta and Jbeta cleavages and rearrangements occurred in DP, rather than in DN, thymocytes; and (iii) most of the 3' Dbeta1 cleavages were coupled to 5' Dbeta2 cleavages instead of to Jbeta cleavages, resulting in nonstandard Dbeta1-Dbeta2-Jbeta2 joints. These findings suggest that the Ebeta regulates TCRbeta rearrangement by promoting accessibility of Dbeta and Jbeta gene segments in DN thymocytes and proper pairing between Dbeta1 and Jbeta gene segments for cleavage and joining in DP thymocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2235807100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC263837PMC
November 2003

Small interfering RNA-mediated gene silencing in T lymphocytes.

J Immunol 2002 Nov;169(10):5754-60

Center for Cancer Research, Massachusetts Institute of Technology, 40 Ames Street E17-526, Cambridge, MA 02139, USA.

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8alpha specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.169.10.5754DOI Listing
November 2002

Erythrocyte-rosetting properties of feline blood lymphocytes and their relationship to monoclonal antibodies to T lymphocytes.

Exp Biol Med (Maywood) 2002 Oct;227(9):771-8

Department of Surgery, University of Tennessee Graduate School of Medicine, Knoxville 37920, USA.

Rosette formation of feline peripheral blood leukocytes with guinea pig (GP) and gerbil (G) erythrocytes (E) has been shown in an earlier study to identify T lymphocytes expressing helper and suppressor cell activity, respectively. This T lymphocyte distinction was based on the removal of the E-rosetting populations from peripheral blood leukocytes (PBL) and the subsequent functional evaluation of the remaining cells in a pokeweed mitogen (PWM)-induced synthesis of immunoglobulin (Ig). In the present study, we demonstrate a direct helper and suppressor function of GPE- and GE-rosetted cells, respectively, wherein the induction of Ig synthesis is altered in a positive or negative way by the addition of the cells to a control target population. A pan-T monoclonal antibody (mAb), CT843, and mAbs to the CD4 (CT248) and CD8 (CT87) subsets are also described; their specificities are established in functional assays, the PWM-induced Ig synthesis and the production of interleukin-2 following Concanavalin A stimulation of PBL, and a biochemical analysis of the surface membrane antigens detected by the mAbs. Immunoprecipitation and SDS-PAGE analyses showed CT248 to react with a approximately 60-kDa protein under both reducing and nonreducing conditions. Under reducing conditions, CT87 reacted with one subunit at approximately 35 kDa; a second faint band at approximately 39 kDa was poorly resolved. mAb CT843 detected a heterodimer of approximately 70 and approximately 60 kDa under both reducing and nonreducing conditions. The relationship of the mAbs to E-rosetting was examined in FACScan analyses and rosette inhibition studies. The percentage of GE-rosetting cells agreed with the percentage of cells stained with the CD8 mAb, whereas a comparison of GPE-rosetting and staining with the CD4 mAb showed variability. The binding of GE to PBL was blocked by pretreatment of PBL with the CD8 mAb, whereas no inhibition of GPE rosettes was observed with any of the mAbs. In a previous study, we had shown that an overnight culture of feline PBL at 37 degrees C leads to the development of a second population of GPE-rosetting cells, also having a helper function. The relationship of the two GPE-rosetting populations to the CD4 mAb, CT248, was examined in rosette depletion studies and FACScan analyses. It was found that depletion of the GPE-rosetting cells from fresh, i.e., Day 0 cells, removed only a small percentage of cells reactive with the CD4 mAb, whereas GPE-rosette depletions performed on Day 1 PBL, which contained both populations of GPE-rosetting cells, removed almost all cells reactive with this antibody. The latter study suggests that the GPE-rosetting phenomenon is detecting two subsets of CD4 cells with T helper function, those present in fresh blood and those acquiring the GPE receptor after an overnight culture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/153537020222700908DOI Listing
October 2002