Showing papers by "Per A. Peterson published in 1999"

TCR-mediated internalization of peptide-MHC complexes acquired by T cells

Abstract: Peptide–major histocompatibility complex protein complexes (pMHCs) on antigen-presenting cells (APCs) are central to T cell activation. Within minutes of peptide-specific T cells interacting with APCs, pMHCs on APCs formed clusters at the site of T cell contact. Thereafter, these clusters were acquired by T cells and internalized through T cell receptor–mediated endocytosis. During this process, T cells became sensitive to peptide-specific lysis by neighboring T cells (fratricide). This form of immunoregulation could explain the “exhaustion” of T cell responses that is induced by high viral loads and may serve to down-regulate immune responses.

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

Abstract: We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

Impaired Immunoproteasome Assembly and Immune Responses in PA28−/− Mice

Abstract: In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

Defective Interleukin (IL)-18–mediated Natural Killer and T Helper Cell Type 1 Responses in IL-1 Receptor–associated Kinase (IRAK)-deficient Mice

Abstract: Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor–associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-κB. The role of IRAK in IL-18–induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-κB activation were found in IRAK-deficient Th1 cells, resulting in a dramatic decrease in interferon (IFN)-γ mRNA expression. In vivo Th1 response to Propionibacterium acnes and lipopolysaccharide in IFN-γ production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-γ production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important role in IL-18–induced signaling and function.

Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: Implications for cellular iron homeostasis

Abstract: Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Recent demonstration of an association between transferrin receptor (TfR) and HFE, a major histocompatibility complex class I-like molecule that has been implicated to play a role in hereditary hemochromatosis, further strengthens the notion that HFE is involved in iron metabolism. Herein we show that TfR is required for and controls the assembly and the intracellular transport and surface expression of HFE. Because surface-expressed HFE and TfR remain firmly associated physically, only the fraction of TfR that is associated with HFE during biosynthesis is affected functionally. Moreover, we show that HFE binding reduces the number of functional transferrin binding sites and impairs TfR internalization, thus reducing the uptake of transferrin-bound iron. Thus, iron homeostasis is indirectly regulated by HFE, a negative modulator of TfR.

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells

Abstract: Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.

Adoptive immunotherapy prevents prostate cancer in a transgenic animal model.

Abstract: Cancer-related mortality can be decreased by prevention, early detection and improved therapies. Although animal models should be used to evaluate the success of cancer therapies, their usefulness is controversial. Many cancer therapies that have cured tumors in mice have not met with similar success when attempted in humans. Current animal models rely mainly on inoculating cell lines into animals, a method that does not reproduce the natural development of the tumor, both for the kinetics of induction and the anatomical site concerned. In this study, we have used an SV40 T-antigen-transgenic mouse model of prostate cancer in which the tumor spontaneously develops orthotopically with a disease progression that closely resembles the progression of human prostate cancer. We have used this model to test the suitability of adoptive cellular immunotherapy. Transfer of naive cells obtained from a T-antigen-negative congenic animal had significant but partial effects: it prevented development of malignant tumors, leaving just minor foci of residual tumor and/or hyperplasia. Adoptive transfer of memory lymphocytes specific for T-antigen, which is a prostatic self antigen in this model, prevented tumor development and progression without affecting the morphology and function of involved tissues. Treated animals were able to breed, and their survival was greatly increased. These results strongly suggest that adoptive immunotherapy should be successful in treating early stages of human prostate cancer.

Cell surface expression of HLA‐E: interaction with human β2‐microglobulin and allelic differences

Abstract: The formation of a trimeric complex composed of MHC class I heavy chain, beta2-microglobulin (beta2m) and peptide ligand is a prerequisite for its efficient transport to the cell surface. We have previously demonstrated impaired intracellular transport of the human class Ib molecule HLA-E in mouse myeloma X63 cells cotransfected with the genes for HLA-E and human beta2m (hbeta2m), which is most likely attributable to inefficient intracellular peptide loading of the HLA-E molecule. Here we demonstrate that cell surface expression of HLA-E in mouse cells strictly depends on the coexpression of hbeta2m and that soluble empty complexes of HLA-E and hbeta2m display a low degree of thermostability. Both observations imply that low affinity interaction of HLA-E with beta2m accounts to a considerable extent for the observed low degree of peptide uptake in the endoplasmic reticulum. Moreover, we show that the only allelic variation present in the caucasoid population located at amino acid position 107 (Gly or Arg) greatly affects intracellular transport and cell surface expression upon transfection of the respective alleles into mouse cells. No obvious difference was found with regard to the sequence of the peptide ligand.

Intercellular adhesion molecule-1 inhibits interleukin 4 production by naive T cells

Abstract: The type of cytokines produced during T cell responses determines susceptibility or resistance to many pathogens and influences the development of autoimmunity and allergy. To define the role of individual accessory molecules in cytokine production during primary immune responses, Drosophila cell lines expressing murine major histocompatibility complex class II molecules with defined combinations of accessory molecules were used to present peptide antigen to naive T cell receptor transgenic T cells. Significantly, expression of B7.1 or B7.2 without additional accessory molecules led to very high production of interleukin (IL)-4, which contrasted with minimal IL-4 production elicited by conventional antigen presenting cells (APC). However, coexpression of ICAM-1 and B7 on Drosophila APC induced little IL-4, suggesting an inhibitory role for intercellular adhesion molecule-1 (ICAM-1). In support of this idea, stimulation of T cell receptor transgenic T cells with peptide presented by splenic APC devoid of ICAM-1 (from ICAM-1-deficient mice) led to high IL-4 production. Thus, the level of IL-4 production by naive CD4+ T cells during typical primary responses appears to be controlled, at least in part, by T–APC interactions involving ICAM-1.

Immune-Associated Nucleotide-1 (IAN-1) Is a Thymic Selection Marker and Defines a Novel Gene Family Conserved in Plants

Abstract: Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.

The role of H2-O and HLA-DO in major histocompatibility complex class II-restricted antigen processing and presentation

Abstract: The function of major histocompatibility complex (MHC) class II molecules is to sample exogenous antigens for presentation to CD4+ T helper cells. After synthesis in the endoplasmic reticulum, class II molecules are directed into the endosomal system by association with the invariant chain (Ii), which is sequentially cleaved, generating class II dimers loaded with Ii-derived peptides (CLIP). These class II-peptide complexes are physiological substrates for H2-M/HLA-DM, a resident of the endosomal/lysosomal system which facilitates the removal of CLIP from newly synthesised class II alpha beta dimers. Exchange of CLIP for antigenic class II-binding peptides is also promoted by the action of H2-M/HLA-DM, resulting in stable peptide-class II complexes that are transported to the cell surface for presentation to CD4+ T cells. Recent evidence suggests that this H2-M/HLA-DM-mediated 'peptide editing' is influenced by another MHC class II-encoded molecule, H2-O/HLA-DO. This non-polymorphic alpha beta heterodimer is associated with H2-M/HLA-DM during intracellular transport and within the endosomal system of B cells. H2-O/HLA-DO alters the peptide exchange function of H2-M/HLA-DM in a pH-dependent manner, so that H2-M/HLA-DM activity is limited to more acidic conditions, corresponding to lysosomal compartments. Indeed, H2-O/HLA-DO may serve to limit the presentation of antigens after fluid phase uptake by B cells, while augmenting presentation of antigens internalised via membrane Ig receptors. Such a mechanism may maintain the fidelity of the B-cell-CD4+ T-cell interaction, counteracting self reactivity arising from less stringent lymphocyte activation. Here, data evaluating the role of H2-O/HLA-DO shall be reviewed and its putative function discussed.

Characterization of the mouse proteasome regulator PA28b gene.

Abstract: The proteasome regulator PA28, which can be upregulated by IFN, is important in the modulation of proteasome activity. Since the proteasome has been implicated in the processing of the major histocompatibility complex (MHC) class I antigens, it was of interest to determine the regulatory elements of PA28 at the genomic level. Although PA28 has been found in different species, the gene layout on the chromosome was not determined. In this study, the genetic organization of mouse PA28b was characterized. Two copies of the PA28b gene, namely b1 and b2, were found by restriction fragment mapping and Southern hybridization. By fluorescence in situ hybridization, the location of the two PA28b genes was determined on chromosomes 11 and 14. PA28b1 has 11 exons, whereas PA28b2 has no introns and appears to be a nonfunctional pseudogene. The 5' promoter region of PA28b1 contains several transcriptional factor binding sites including two IFN responsive elements. The expression levels of PA28 and other gene products involved in MHC class I antigen presentation appear to be correlated in various tissues. Notably, PA28 is expressed at high levels in immunological tissues such as spleen and peripheral blood leukocytes. Taken together, PA28 seems to be co-regulated with other molecules involved in MHC class I antigen presentation.

A Fluorescence-Based High Throughput Screen for the Transporter Associated with Antigen Processing

Abstract: The transporter associated with antigen processing (TAP) is essential for antigen presentation by major histocompatibility complex (MHC) class I molecules. Traditional methods used to analyze peptide transport mediated by TAP require radioactive labeling of peptides and time-consuming manipulation of Concanavalin A-Sepharose. Drug discovery research requires rapid and reliable evaluation of large number of samples for bioactivity. To meet these requirements a nonradioactive, HTS assay for peptide transport activity of TAP has been developed. The radioactive label in the traditional assays has been replaced by a fluorescent label without compromising the transport efficiency of labeled peptide or the sensitivity of the assay. The use of multiscreen filtration plates has facilitated higher throughput and eliminated the centrifugation steps used in traditional TAP assays. The HTS assay shows similar kinetic characteristics as compared to the traditional assay. The HTS assay has been adapted on a Quadratrade mark 96-320 96-channel pipetting station (Tomtec, Hamden, CT) by optimizing time course, dose response of TAP to peptides and adenosine triphosphate (ATP), signal/noise ratio, reproducibility, and reagent stability. This HTS system has been utilized to screen a multiplexed compound library with a maximum of throughput 17,600 compounds per week.