Publications by authors named "Etienne Gagnon"

38 Publications

CAMAP: Artificial neural networks unveil the role of codon arrangement in modulating MHC-I peptides presentation.

PLoS Comput Biol 2021 Oct 22;17(10):e1009482. Epub 2021 Oct 22.

Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Canada.

MHC-I associated peptides (MAPs) play a central role in the elimination of virus-infected and neoplastic cells by CD8 T cells. However, accurately predicting the MAP repertoire remains difficult, because only a fraction of the transcriptome generates MAPs. In this study, we investigated whether codon arrangement (usage and placement) regulates MAP biogenesis. We developed an artificial neural network called Codon Arrangement MAP Predictor (CAMAP), predicting MAP presentation solely from mRNA sequences flanking the MAP-coding codons (MCCs), while excluding the MCC per se. CAMAP predictions were significantly more accurate when using original codon sequences than shuffled codon sequences which reflect amino acid usage. Furthermore, predictions were independent of mRNA expression and MAP binding affinity to MHC-I molecules and applied to several cell types and species. Combining MAP ligand scores, transcript expression level and CAMAP scores was particularly useful to increase MAP prediction accuracy. Using an in vitro assay, we showed that varying the synonymous codons in the regions flanking the MCCs (without changing the amino acid sequence) resulted in significant modulation of MAP presentation at the cell surface. Taken together, our results demonstrate the role of codon arrangement in the regulation of MAP presentation and support integration of both translational and post-translational events in predictive algorithms to ameliorate modeling of the immunopeptidome.
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http://dx.doi.org/10.1371/journal.pcbi.1009482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577786PMC
October 2021

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Mol Cell 2021 09;81(18):3848-3865.e19

CRCHUM, 900 Saint-Denis St, Montréal, QC H2X 0A9, Canada; Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada. Electronic address:

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.
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http://dx.doi.org/10.1016/j.molcel.2021.08.028DOI Listing
September 2021

CD40L-Stimulated B Lymphocytes Are Polarized toward APC Functions after Exposure to IL-4 and IL-21.

J Immunol 2021 Jun 16. Epub 2021 Jun 16.

Axe Cancer, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Québec, Canada;

B lymphocytes have multiple functions central to humoral immunity, including Ag presentation to T cells, cytokine secretion, and differentiation into Ab-secreting plasma cells. In vitro expansion of human B cells by continuous IL-4 stimulation and engagement of their CD40 receptor by CD40L has allowed the use of these IL-4-CD40-B cells in research for the induction of Ag-specific T cell immune responses. However, in vivo, follicular helper T cells also influence B cell activity through the secretion of IL-21. The impact of both cytokines on multiple B cell functions is not clearly defined. To further understand these cytokines in CD40-B cell biology, we stimulated CD40-B cells with IL-4 or IL-21 or both (Combo) and characterized the proliferation, subsets, and functions of these cells. We demonstrate that IL-21- and Combo-CD40-B cells are highly proliferative cells that can be rapidly expanded to high numbers. We show that IL-21-CD40-B cells polarize to Ab-secreting plasma cells, whereas IL-4- and Combo-CD40-B cells are mostly activated mature B cells that express molecules associated with favorable APC functions. We further demonstrate that both IL-4- and Combo-CD40-B cells are efficient in promoting T cell activation and proliferation compared with IL-21-CD40-B cells. Thus, our study provides a better appreciation of CD40-B cell plasticity and biology. In addition, the stimulation of B cells with CD40L, IL-4, and IL-21 allows for the fast generation of high numbers of efficient APC, therefore providing a prospective tool for research and clinical applications such as cancer immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.2001173DOI Listing
June 2021

TMEM16F mediates bystander TCR-CD3 membrane dissociation at the immunological synapse and potentiates T cell activation.

Sci Signal 2021 03 23;14(675). Epub 2021 Mar 23.

Institut de Recherche en Immunologie et Cancérologie, 2950 Chemin de la Polytechnique, Montréal, Québec H3T1J4, Canada.

Electrostatic interactions regulate many aspects of T cell receptor (TCR) activity, including enabling the dynamic binding of the TCR-associated CD3ε and CD3ζ chains to anionic lipids in the plasma membrane to prevent spontaneous phosphorylation. Substantial changes in the electrostatic potential of the plasma membrane occur at the immunological synapse, the interface between a T cell and an antigen-presenting cell. Here, we investigated how the electrostatic interactions that promote dynamic membrane binding of the TCR-CD3 cytoplasmic domains are modulated during signaling and affect T cell activation. We found that Ca-dependent activation of the phosphatidylserine scramblase TMEM16F, which was previously implicated in T cell activation, reduced the electrostatic potential of the plasma membrane during immunological synapse formation by locally redistributing phosphatidylserine. This, in turn, increased the dissociation of bystander TCR-CD3 cytoplasmic domains from the plasma membrane and enhanced TCR-dependent signaling and consequently T cell activation. This study establishes the molecular basis for the role of TMEM16F in bystander TCR-induced signal amplification and identifies enhancement of TMEM16F function as a potential therapeutic strategy for promoting T cell activation.
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http://dx.doi.org/10.1126/scisignal.abb5146DOI Listing
March 2021

Critical role for TRIM28 and HP1β/γ in the epigenetic control of T cell metabolic reprograming and effector differentiation.

Proc Natl Acad Sci U S A 2019 12 27;116(51):25839-25849. Epub 2019 Nov 27.

Institut Curie, Université Paris Sciences et Lettres, INSERM U932, 75005 Paris, France;

Naive CD4 T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4 T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 β and γ isoforms (HP1β/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28 T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28 regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1β/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.
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http://dx.doi.org/10.1073/pnas.1901639116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925996PMC
December 2019

Electrostatic interactions: From immune receptor assembly to signaling.

Immunol Rev 2019 09;291(1):26-43

Institut de Recherche en Immunologie et Cancérologie/Institute for Research in Immunology and Cancer, Montréal, Québec, Canada.

Our ability to mount a long-lasting and protective immune response relies on a variety of immune receptors that enable the recognition of ongoing infections, which triggers the adaptation of a myriad of immune cells. The organization of several immune receptors, such as the T cells receptor and several natural killer cell receptors, utilizes different modules for ligand recognition and signaling. These receptors require specific recognition mechanisms between the different modules in order to ensure proper assembly and function. Once assembled, immune receptors must remain inactive in the absence of ligand to prevent the onset of unwanted immune response. Indeed, several mechanisms exist to prevent aberrant immune receptor signaling in the absence of ligand to avert the initiation of uncontrolled autoimmunity. However, once a ligand is recognized, immune receptors must rapidly and specifically engage kinases to initiate highly regulated signaling cascades that lead to the initiation of transcriptional programs that dictate the immune response. Over the last decade, compelling evidence have been presented which suggest that electrostatic interactions are critical for many aspects of immune receptor functions. In the work that follows, we present an overview of the literature that have provided evidence that illustrate how electrostatic interactions regulate immune receptor assembly, inactive state, triggering, and signaling.
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http://dx.doi.org/10.1111/imr.12769DOI Listing
September 2019

The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole.

PLoS Pathog 2019 07 29;15(7):e1007982. Epub 2019 Jul 29.

INRS, Centre Armand-Frappier Santé Biotechnologie, Laval, Québec, Canada.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.
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http://dx.doi.org/10.1371/journal.ppat.1007982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687203PMC
July 2019

The sequence features that define efficient and specific hAGO2-dependent miRNA silencing guides.

Nucleic Acids Res 2018 09;46(16):8181-8196

Institut de recherche en immunologie et en cancérologie (IRIC), Université de Montréal, Montréal, Québec H3C 3J7, Canada.

MicroRNAs (miRNAs) are ribonucleic acids (RNAs) of ∼21 nucleotides that interfere with the translation of messenger RNAs (mRNAs) and play significant roles in development and diseases. In bilaterian animals, the specificity of miRNA targeting is determined by sequence complementarity involving the seed. However, the role of the remaining nucleotides (non-seed) is only vaguely defined, impacting negatively on our ability to efficiently use miRNAs exogenously to control gene expression. Here, using reporter assays, we deciphered the role of the base pairs formed between the non-seed region and target mRNA. We used molecular modeling to reveal that this mechanism corresponds to the formation of base pairs mediated by ordered motions of the miRNA-induced silencing complex. Subsequently, we developed an algorithm based on this distinctive recognition to predict from sequence the levels of mRNA downregulation with high accuracy (r2 > 0.5, P-value < 10-12). Overall, our discovery improves the design of miRNA-guide sequences used to simultaneously downregulate the expression of multiple predetermined target genes.
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http://dx.doi.org/10.1093/nar/gky546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6144789PMC
September 2018

Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

Proc Natl Acad Sci U S A 2018 03 27;115(11):E2653-E2662. Epub 2018 Feb 27.

Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC H3T 1J4, Canada;

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.
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http://dx.doi.org/10.1073/pnas.1716224115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856531PMC
March 2018

Detection of Quiescent Radioresistant Epithelial Progenitors in the Adult Thymus.

Front Immunol 2017 5;8:1717. Epub 2017 Dec 5.

Institute for Research in Immunology and Cancer, Montreal, QC, Canada.

Thymic aging precedes that of other organs and is initiated by the gradual loss of thymic epithelial cells (TECs). Based on culture and transplantation assays, recent studies have reported on the presence of thymic epithelial progenitor cells (TEPCs) in young adult mice. However, the physiological role and properties of TEPC populations reported to date remain unclear. Using an label-retention assay, we previously identified a population of quiescent but non-senescent TECs. The goals of this study were therefore (i) to evaluate the contribution of these quiescent TECs to thymic regeneration following irradiation-induced acute thymic injury and (ii) to characterize their phenotypic and molecular profiles using flow cytometry, immunohistology, and transcriptome sequencing. We report that while UEA1 cells cycle the most in steady state, they are greatly affected by irradiation, leading to cell loss and proliferative arrest following acute thymic involution. On the opposite, the UEA1 subset of quiescent TECs is radioresistant and proliferate following acute thymic involution, thereby contributing to thymic regeneration in 28- to 30-week-old mice. UEA1 quiescent TECs display an undifferentiated phenotype (co-expression of K8 and K5 cytokeratins) and express high levels of genes that regulate stem cell activity in different tissues (e.g., and ). In addition, two features suggest that UEA1 quiescent TECs occupy discrete stromal niches: (i) their preferential location in clusters adjacent to the cortico-medullary junction and (ii) their high expression of genes involved in cross talk with mesenchymal cells. The ability of UEA1 quiescent TECs to participate to TEC regeneration qualifies them as progenitor cells particularly relevant in the context of regeneration following acute thymic injury.
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http://dx.doi.org/10.3389/fimmu.2017.01717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723310PMC
December 2017

Studying Dynamic Plasma Membrane Binding of TCR-CD3 Chains During Immunological Synapse Formation Using Donor-Quenching FRET and FLIM-FRET.

Methods Mol Biol 2017 ;1584:259-289

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02115, USA.

Over the last decade, advancements in the time and space resolution of microscopy technologies have enabled dissection of the molecular events involved in T cell Immunological Synapse (IS) formation. Using a combination of Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imagining Microscopy (FLIM), we have demonstrated dynamic plasma membrane binding by cytoplasmic domains of T cell receptor (TCR)-associated CD3 chains and other T cell transmembrane receptors. We have developed methods for imaging such membrane binding both at steady state and during receptor triggering at the IS. Plasma membrane binding by cytoplasmic domains may represent a novel mechanism for regulating the signaling function of important receptors in the immune system.
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http://dx.doi.org/10.1007/978-1-4939-6881-7_16DOI Listing
February 2018

Binding of the cytoplasmic domain of CD28 to the plasma membrane inhibits Lck recruitment and signaling.

Sci Signal 2016 07 26;9(438):ra75. Epub 2016 Jul 26.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. Program in Immunology, Harvard Medical School, Boston, MA 02115, USA. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.

The T cell costimulatory receptor CD28 is required for the full activation of naïve T cells and for the development and maintenance of Foxp3(+) regulatory T (Treg) cells. We showed that the cytoplasmic domain of CD28 was bound to the plasma membrane in resting cells and that ligand binding to CD28 resulted in its release. Membrane binding by the CD28 cytoplasmic domain required two clusters of basic amino acid residues, which interacted with the negatively charged inner leaflet of the plasma membrane. These same clusters of basic residues also served as interaction sites for Lck, a Src family kinase critical for CD28 function. This signaling complex was further stabilized by the Lck-mediated phosphorylation of CD28 Tyr(207) and the subsequent binding of the Src homology 2 (SH2) domain of Lck to this phosphorylated tyrosine. Mutation of the basic clusters in the CD28 cytoplasmic domain reduced the recruitment to the CD28-Lck complex of protein kinase Cθ (PKCθ), which serves as a key effector kinase in the CD28 signaling pathway. Consequently, mutation of either a basic cluster or Tyr(207) impaired CD28 function in mice as shown by the reduced thymic differentiation of FoxP3(+) Treg cells. On the basis of these results, we propose a previously undescribed model for the initiation of CD28 signaling.
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http://dx.doi.org/10.1126/scisignal.aaf0626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929992PMC
July 2016

Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation.

Cell 2016 Jul 23;166(2):314-327. Epub 2016 Jun 23.

Département de Pathologie et Biologie Cellulaire, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, QC H3C 3J7, Canada. Electronic address:

Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology.
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http://dx.doi.org/10.1016/j.cell.2016.05.039DOI Listing
July 2016

Membrane association of the CD3ε signaling domain is required for optimal T cell development and function.

J Immunol 2014 Jul 4;193(1):258-67. Epub 2014 Jun 4.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105;

The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.
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http://dx.doi.org/10.4049/jimmunol.1400322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065803PMC
July 2014

Leishmania evades host immunity by inhibiting antigen cross-presentation through direct cleavage of the SNARE VAMP8.

Cell Host Microbe 2013 Jul;14(1):15-25

Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, QC H3C 3J7, Canada.

During phagocytosis, microorganisms are taken up by immune cells into phagosomes. Through membrane-trafficking events mediated by SNARE proteins, phagosomes fuse with lysosomes, generating degradative phagolysosomes. Phagolysosomes contribute to host immunity by linking microbial killing within these organelles with antigen processing for presentation on MHC class I or II molecules to T cells. We show that the intracellular parasite Leishmania evades immune recognition by inhibiting phagolysosome biogenesis. The Leishmania cell surface metalloprotease GP63 cleaves a subset of SNAREs, including VAMP8. GP63-mediated VAMP8 inactivation or Vamp8 disruption prevents the NADPH oxidase complex from assembling on phagosomes, thus altering their pH and degradative properties. Consequently, the presentation of exogenous Leishmania antigens on MHC class I molecules, also known as cross-presentation, is inhibited, resulting in reduced T cell activation. These findings indicate that Leishmania subverts immune recognition by altering phagosome function and highlight the importance of VAMP8 in phagosome biogenesis and antigen cross-presentation.
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http://dx.doi.org/10.1016/j.chom.2013.06.003DOI Listing
July 2013

Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain.

J Exp Med 2012 Dec 19;209(13):2423-39. Epub 2012 Nov 19.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide--MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.
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http://dx.doi.org/10.1084/jem.20120790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526357PMC
December 2012

β-Selection-induced proliferation is required for αβ T cell differentiation.

Immunity 2012 Nov;37(5):840-53

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

Proliferation and differentiation are tightly coordinated to produce an appropriate number of differentiated cells and often exhibit an antagonistic relationship. Developing T cells, which arise in the thymus from a minute number of bone-marrow-derived progenitors, undergo a major expansion upon pre-T cell receptor (TCR) expression. The burst of proliferation coincides with differentiation toward the αβ T cell lineage-but the two processes were previously thought to be independent from one another, although both were driven by signaling from pre-TCR and Notch receptors. Here we report that proliferation at this step was not only absolutely required for differentiation but also that its ectopic activation was sufficient to substantially rescue differentiation in the absence of Notch signaling. Consistently, pharmacological inhibition of the cell cycle machinery also blocked differentiation in vivo. Thus the proliferation step is strictly required prior to differentiation of immature thymocytes.
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http://dx.doi.org/10.1016/j.immuni.2012.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709258PMC
November 2012

Alternative RISC assembly: binding and repression of microRNA-mRNA duplexes by human Ago proteins.

RNA 2012 Nov 27;18(11):2041-55. Epub 2012 Sep 27.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to the consensus view that all miRNAs are associated with Argonaute (Ago) proteins, we determine that miRNAs are expressed in a 13-fold excess relative to Agos in HeLa cells and that miRNAs are bound to mRNAs in a sevenfold excess relative to Agos, implying the existence of miRNA-mRNA duplexes not stoichiometrically bound by Agos. We show that all four human Agos can repress miRNA-mRNA duplexes, but only Ago2 can cleave small interfering RNA-mRNA duplexes in vitro. We visualize direct Ago binding to miRNA-mRNA duplexes in live cells using fluorescence lifetime imaging microscopy. In contrast to the consensus view that Agos bind miRNA duplexes, these data demonstrate that Agos can bind and repress miRNA-mRNA duplexes and support a model of catalytic Ago function in translational repression.
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http://dx.doi.org/10.1261/rna.035675.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479394PMC
November 2012

Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome.

Mol Cell Proteomics 2012 Jul 15;11(7):M111.016378. Epub 2012 Mar 15.

Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Quebec, H3C 3J7, Canada.

Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.
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http://dx.doi.org/10.1074/mcp.M111.016378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394953PMC
July 2012

Self-reactive human CD4 T cell clones form unusual immunological synapses.

J Exp Med 2012 Feb 6;209(2):335-52. Epub 2012 Feb 6.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

Recognition of self-peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR-pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.
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http://dx.doi.org/10.1084/jem.20111485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280872PMC
February 2012

The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity.

Biochem Biophys Res Commun 2012 Feb 28;418(4):786-91. Epub 2012 Jan 28.

Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA 02215, United States.

Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class I and related genes. While a 'master regulator' of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine.
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http://dx.doi.org/10.1016/j.bbrc.2012.01.104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289513PMC
February 2012

Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling.

Cold Spring Harb Perspect Biol 2010 Apr 17;2(4):a005140. Epub 2010 Mar 17.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

The T-cell receptor (TCR)-CD3 complex serves as a central paradigm for general principles of receptor assembly, ligand recognition, and signaling in the immune system. There is no other receptor system that matches the diversity of both receptor and ligand components. The recent expansion of the immunological structural database is beginning to identify key principles of MHC and peptide recognition. The multicomponent assembly of the TCR complex illustrates general principles used by many receptors in the immune system, which rely on basic and acidic transmembrane residues to guide assembly. The intrinsic binding of the cytoplasmic domains of the CD3epsilon and zeta chains to the inner leaflet of the plasma membrane represents a novel mechanism for control of receptor activation: Insertion of critical CD3epsilon tyrosines into the hydrophobic membrane core prevents their phosphorylation before receptor engagement.
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http://dx.doi.org/10.1101/cshperspect.a005140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845206PMC
April 2010

Positively selecting peptides: their job does not end in the thymus.

Nat Immunol 2009 Nov;10(11):1143-4

Peptides able to positively select major histocompatibility complex class II-restricted thymocytes have not yet been defined. Two new reports identify and ascribe important extrathymic functions to several positively selecting peptides for CD4+ T cells.
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http://dx.doi.org/10.1038/ni1109-1143DOI Listing
November 2009

Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.

Cell 2008 Nov;135(4):702-13

Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.
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http://dx.doi.org/10.1016/j.cell.2008.09.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597348PMC
November 2008

Observing the creation of electronic feshbach resonances in soft x-ray-induced O2 dissociation.

Science 2008 Nov;322(5904):1081-5

JILA, University of Colorado at Boulder, Boulder, CO 80309, USA.

When an atom or molecule is ionized by an x-ray, highly excited states can be created that then decay, or autoionize, by ejecting a second electron from the ion. We found that autoionization after soft x-ray photoionization of molecular oxygen follows a complex multistep process. By interrupting the autoionization process with a short laser pulse, we showed that autoionization cannot occur until the internuclear separation of the fragments is greater than approximately 30 angstroms. As the ion and excited neutral atom separated, we directly observed the transformation of electronically bound states of the molecular ion into Feshbach resonances of the neutral oxygen atom that are characterized by both positive and negative binding energies. States with negative binding energies have not previously been predicted or observed in neutral atoms.
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http://dx.doi.org/10.1126/science.1164498DOI Listing
November 2008

Direct measurement of the angular dependence of the single-photon ionization of aligned N2 and CO2.

J Phys Chem A 2008 Oct 12;112(39):9382-6. Epub 2008 Aug 12.

JILA and Department of Physics, University of Colorado and NIST, Boulder, Colorado 80309, USA.

By combining a state-of-the-art high-harmonic ultrafast soft X-ray source with field-free dynamic alignment, we map the angular dependence of molecular photoionization yields for the first time for a nondissociative molecule. The observed modulation in ion yield as a function of molecular alignment is attributed to the molecular frame transition dipole moment of single-photon ionization to the X, A and B states of N2(+) and CO2(+). Our data show that the transition dipoles for single-photon ionization of N2 and CO2 at 43 eV have larger perpendicular components than parallel ones. A direct comparison with published theoretical partial wave ionization cross-sections confirms these experimental observations, which are the first results to allow such comparison with theory for bound cation states. The results provide the first step toward a novel method for measuring molecular frame transition dipole matrix elements.
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http://dx.doi.org/10.1021/jp8023414DOI Listing
October 2008

Time-resolved momentum imaging system for molecular dynamics studies using a tabletop ultrafast extreme-ultraviolet light source.

Rev Sci Instrum 2008 Jun;79(6):063102

JILA, University of Colorado at Boulder, Boulder, CO 80309-0440, USA.

We describe a momentum imaging setup for direct time-resolved studies of ionization-induced molecular dynamics. This system uses a tabletop ultrafast extreme-ultraviolet (EUV) light source based on high harmonic upconversion of a femtosecond laser. The high photon energy (around 42 eV) allows access to inner-valence states of a variety of small molecules via single photon excitation, while the sub--10-fs pulse duration makes it possible to follow the resulting dynamics in real time. To obtain a complete picture of molecular dynamics following EUV induced photofragmentation, we apply the versatile cold target recoil ion momentum spectroscopy reaction microscope technique, which makes use of coincident three-dimensional momentum imaging of fragments resulting from photoexcitation. This system is capable of pump-probe spectroscopy by using a combination of EUV and IR laser pulses with either beam as a pump or probe pulse. We report several experiments performed using this system.
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http://dx.doi.org/10.1063/1.2930869DOI Listing
June 2008

Soft X-ray-driven femtosecond molecular dynamics.

Science 2007 Sep;317(5843):1374-8

JILA, University of Colorado, Boulder, CO 80309-0441, USA.

The direct observation of molecular dynamics initiated by x-rays has been hindered to date by the lack of bright femtosecond sources of short-wavelength light. We used soft x-ray beams generated by high-harmonic upconversion of a femtosecond laser to photoionize a nitrogen molecule, creating highly excited molecular cations. A strong infrared pulse was then used to probe the ultrafast electronic and nuclear dynamics as the molecule exploded. We found that substantial fragmentation occurs through an electron-shakeup process, in which a second electron is simultaneously excited during the soft x-ray photoionization process. During fragmentation, the molecular potential seen by the electron changes rapidly from nearly spherically symmetric to a two-center molecular potential. Our approach can capture in real time and with angstrom resolution the influence of ionizing radiation on a range of molecular systems, probing dynamics that are inaccessible with the use of other techniques.
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http://dx.doi.org/10.1126/science.1144920DOI Listing
September 2007

Multi-kilohertz repetition rate Ti:sapphire amplifier based on down-chirped pulse amplification.

Opt Express 2006 Oct;14(20):9277-83

We present a novel ultrafast multipass laser amplifier design optimized for sub-millijoule output energy and capable of being operated at repetition rates exceeding 40 kHz. This ti:sapphire based system makes use of a grism based stretcher, a cryogenically cooled ti:sapphire crystal and an astigmatically compensated multipass amplifier design that allows for pumping with significantly lower pump pulse energies than has been demonstrated to date. We also make use of the downchirped pulse amplification scheme to minimize loss in the pulse compression process. Preliminary experiments demonstrate an output pulse energy of 290 muJ at 10 kHz and 270 muJ at 15 kHz with a pulse duration of 36 fs.
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http://dx.doi.org/10.1364/oe.14.009277DOI Listing
October 2006
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