Gijsbert M Grotenbreg

Publications of Gijsbert M Grotenbreg

• Engineering T cells specific for a dominant severe acute respiratory syndrome coronavirus CD8 T cell epitope.

  Authors: Hsueh-Ling Janice Oh, Adeline Chia, Cynthia Xin Lei Chang, Hoe Nam Leong, Khoon Lin Ling, Gijsbert M Grotenbreg, Adam J Gehring, Yee Joo Tan, Antonio Bertoletti

  Journal of virology. 08/2011; 85(20):10464-71.

  Severe acute respiratory syndrome (SARS) is a highly contagious and life threatening disease, with a fatality rate of almost 10%. The etiologic agent is a novel coronavirus, severe acute respiratorySevere acute respiratory syndrome (SARS) is a highly contagious and life threatening disease, with a fatality rate of almost 10%. The etiologic agent is a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), with animal reservoirs found in bats and other wild animals and thus the possibility of reemergence. In this study, we first investigated at 6 years postinfection whether SARS-specific memory T cells persist in SARS-recovered individuals, demonstrating that these subjects still possess polyfunctional SARS-specific memory CD4+ and CD8+ T cells. A dominant memory CD8+ T cell response against SARS-CoV nucleocaspid protein (NP; amino acids 216 to 225) was then defined in SARS-recovered individuals carrying HLA-B*40:01, a HLA-B molecule present in approximately one-quarter of subjects of Asian ethnicities. To reconstitute such a CD8+ T cell response, we isolated the alpha and beta T cell receptors of the HLA-B*40:01-restricted SARS-specific CD8+ T cells. Using T cell receptor gene transfer, we generated SARS-specific redirected T cells from the lymphocytes of normal individuals. These engineered CD8+ T cells displayed avidity and functionality similar to that of natural SARS-specific memory CD8+ T cells. They were able to degranulate and produce gamma interferon, tumor necrosis factor alpha, and macrophage inflammatory proteins 1α and 1β after antigenic stimulation. Since there is no effective treatment against SARS, these transduced T cells specific for an immunodominant SARS epitope may provide a new avenue for treatment during a SARS outbreak.

• Stability screening of arrays of major histocompatibility complexes on combinatorially encoded flow cytometry beads.

  Authors: Shi Ling Chew, Ming Yan Or, Cynthia Xin Lei Chang, Adam J Gehring, Antonio Bertoletti, Gijsbert M Grotenbreg

  The Journal of biological chemistry. 06/2011; 286(32):28466-75.

  The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellularThe binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

• Sources of diversity in T cell epitope discovery.

  Authors: Cynthia Xin Lei Chang, Lingyun Dai, Zhen Wei Tan, Joanna Ai Ling Choo, Antonio Bertoletti, Gijsbert M Grotenbreg

  Frontiers in bioscience : a journal and virtual library. 01/2011; 17:3014-35.

  CD8-positive T cells respond to small antigenic peptide fragments presented on class I major histocompatibility complexes (MHCs). Those specific T cell epitopes capable of precipitating a cellularCD8-positive T cells respond to small antigenic peptide fragments presented on class I major histocompatibility complexes (MHCs). Those specific T cell epitopes capable of precipitating a cellular immune response are either derived from (altered) self (i.e. they are autoimmune- or cancer-associated) or come from foreign sources (i.e. they are pathogen-associated). Identification of T cell epitopes provides elementary information that can be employed in technologies that monitor and predict the likely outcome of an immune response, as well as in therapeutic and vaccine development efforts. The coexistence between host and pathogen has largely driven the diversification of both their systems of immune surveillance and their antigenic determinants, respectively. In this review, we discuss the multitude of factors that introduce diversity to the T cell response from both sides of the host-pathogen interaction. Furthermore, we provide an overview of a variety of commonly employed methods and tools to characterize class I MHC restricted antigen presentation and recent endeavors towards the harmonization of reporting data concerning T cell responses.

• Transnuclear mice with predefined T cell receptor specificities against Toxoplasma gondii obtained via SCNT.

  Authors: Oktay Kirak, Eva-Maria Frickel, Gijsbert M Grotenbreg, Heikyung Suh, Rudolf Jaenisch, Hidde L Ploegh

  Science (New York, N.Y.). 04/2010; 328(5975):243-8.

  Mice that are transgenic for rearranged antigen-specific T cell receptors (TCRs) are essential tools to study T cell development and function. Such TCRs are usually isolated from the relevant T cellsMice that are transgenic for rearranged antigen-specific T cell receptors (TCRs) are essential tools to study T cell development and function. Such TCRs are usually isolated from the relevant T cells after long-term culture, often after repeated antigen stimulation, which unavoidably skews the T cell population used. Random genomic integration of the TCR alpha and beta chain and expression from nonendogenous promoters represent additional drawbacks of transgenics. Using epigenetic reprogramming via somatic cell nuclear transfer, we demonstrated that T cells with predefined specificities against Toxoplasma gondii can be used to generate mouse models that express the TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

• Transnuclear mice with pre-defined T cell receptor specificities against Toxoplasma gondii obtained via SCNT.

  Authors: Oktay Kirak, Eva-Maria Frickel, Gijsbert M Grotenbreg, Heikyung Suh, Rudolf Jaenisch, Hidde L Ploegh

  Journal of visualized experiments : JoVE. 01/2010;

  Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have beenLymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR α- and β-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics. Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

• Differential regulation of effector- and central-memory responses to Toxoplasma gondii Infection by IL-12 revealed by tracking of Tgd057-specific CD8+ T cells.

  Authors: Douglas C Wilson, Gijsbert M Grotenbreg, Kenian Liu, Yanlin Zhao, Eva-Maria Frickel, Marc-Jan Gubbels, Hidde L Ploegh, George S Yap

  PLoS pathogens. 01/2010; 6(3):e1000815.

  Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-gamma-producing effector T cells during Toxoplasma gondii infection. However, the role ofProduction of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-gamma-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K(b)-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-gamma production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-gamma recall responses. Importantly, isolated CD62L(hi) KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-gamma-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.

• Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity.

  Authors: John M Antos, Guo-Liang Chew, Carla P Guimaraes, Nicholas C Yoder, Gijsbert M Grotenbreg, Maximilian Wei-Lin Popp, Hidde L Ploegh

  Journal of the American Chemical Society. 08/2009;

  The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide withThe unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.

• Parasite Stage-Specific Recognition of Endogenous Toxoplasma gondii-Derived CD8(+) T Cell Epitopes.

  Authors: Eva-Maria Frickel, Nivedita Sahoo, Johnathan Hopp, Marc-Jan Gubbels, Mary Patricia J Craver, Laura J Knoll, Hidde L Ploegh, Gijsbert M Grotenbreg

  The Journal of infectious diseases. 11/2008;

  Background. @nbsp; BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans.Background. @nbsp; BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8(+) T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. Methods. @nbsp; We employed caged major histocompatibility complex molecules to generate 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8(+) T cells in BALB/c mice. Results. @nbsp; We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Conclusions. @nbsp; Our results shed light on the identity and the parasite stage-specificity of 2 CD8(+) T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.

• The CD8 T cell response against Murine gamma herpes virus (MHV-68) is directed towards a broad repertoire of epitopes from both early and late antigens.

  Authors: Sara Gredmark-Russ, Evelyn J. Cheung, Marisa K Isaacson, Hidde L Ploegh, Gijsbert M Grotenbreg

  Journal of virology. 11/2008;

  Infection of mice with murine gamma herpes virus (MHV-68) robustly activates CD8 T cells, but only six Class I MHC restricted epitopes have been described to date for the widely used H-2(b) haplotypeInfection of mice with murine gamma herpes virus (MHV-68) robustly activates CD8 T cells, but only six Class I MHC restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the CTL response in MHV-68 infected C57BL/6 mice, we screened for H-2K(b) and H-2D(b) restricted epitopes using a set of 384 candidate epitopes in a MHC tetramer based approach, and identified 19 new epitopes in 16 different ORFs. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T cell specific responses for the remaining three. The peak of the CD8 T cell response to most peptides occurs between 6 and 10 days post infection (dpi). The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62(lo) and CD44(hi)) and produce IFN-gamma upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T cell repertoire and more rapid activation than previously recorded.

• Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers.

  Authors: Gijsbert M Grotenbreg, Nadia R Roan, Eduardo Guillen, Rob Meijers, Jia-huai Wang, George W Bell, Michael N Starnbach, Hidde L Ploegh

  Proceedings of the National Academy of Sciences of the United States of America. 04/2008; 105(10):3831-6.

  Class I MHC tetramers allow direct phenotypic identification of CD8(+) T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loadedClass I MHC tetramers allow direct phenotypic identification of CD8(+) T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loaded with conditional ligands (caged MHC molecules) provides a fast and straightforward method to obtain diverse arrays of class I MHC tetramers and facilitates CD8(+) T cell epitope discovery. Here, we describe the development of photocleavable analogs of the FAPGNYPAL (SV9) epitope that bind H-2K(b) and H-2D(b) with full retention of their structural and functional integrity. We ranked all possible H-2K(b) octameric and H-2D(b) nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from approximately 2,000 of the highest scoring peptides by replacement of the SV9 analog with the peptide of choice. The resulting 2,000-member class I MHC tetramer array allowed the discovery of two variants of an epitope derived from polymorphic membrane protein I (PmpI) and an assessment of the kinetics of emergence and the effector function of the corresponding CD8(+) T cells.

• Sortagging: a versatile method for protein labeling.

  Authors: Maximilian W Popp, John M Antos, Gijsbert M Grotenbreg, Eric Spooner, Hidde L Ploegh

  Nature chemical biology. 11/2007; 3(11):707-8.

  Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. SortaggingGenetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the precision of a genetically encoded tag with the specificity of an enzymatic reaction and the ease and chemical versatility of peptide synthesis. Here we apply this technique to proteins in vitro and on the surface of living cells.

• Empty class II major histocompatibility complex created by peptide photolysis establishes the role of DM in peptide association.

  Authors: Gijsbert M Grotenbreg, Melissa J Nicholson, Kevin D Fowler, Kathrin Wilbuer, Leah Octavio, Maxine Yang, Arup K Chakraborty, Hidde L Ploegh, Kai W Wucherpfennig

  The Journal of biological chemistry. 08/2007; 282(29):21425-36.

  DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult toDM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

• A microengraving method for rapid selection of single cells producing antigen-specific antibodies.

  Authors: J Christopher Love, Jehnna L Ronan, Gijsbert M Grotenbreg, Annemarthe G Van der Veen, Hidde L Ploegh

  Nature biotechnology. 07/2006; 24(6):703-7.

  Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval ofMonoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.