Showing papers by "Per A. Peterson published in 1996"

An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex

Abstract: The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the αβ T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated αβ TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Vα and Vβ domains but deviates in the constant Cα domain and in the interdomain pairing of Cα with Cβ. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Cα-Cβ interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the α and β chains. The 2C TCR covers the class I MHC H-2K b binding groove so that the Vα CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vβ chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Vα and Vβ CDR3s straddle the peptide between the helices around the central position of the peptide.

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process

Abstract: The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle.

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

Abstract: The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.

CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes

Abstract: T-cell antigen receptors (TCR) generally interact with moderate affinity with the complex formed by major histocompatibility complex (MHC) molecules and foreign peptides. MHC/TCR recognition is followed by the generation of a signal to the T cell through a monomorphic multicomponent system that includes the CD3 complex and accessory molecules such as CD4 and CD8. The interaction between the extracellular domains of MHC and TCR molecules, and the interaction of MHC and CD4/CD8 molecules, have been considered to occur independently of one another. We report here that the affinity of CD8 dimers for MHC class I molecules is independent of haplotype and peptide content, and that the affinity of the TCR for its specific ligand is enhanced through a reduced 'off' rate in the presence of either CD8alpha alpha homo- or CD8alpha beta heterodimers. Moreover, CD8 seems to help recognition of the specific MHC-peptide complex either by guiding an energetically favourable docking of TCR onto MHC, or by inducing conformational changes in the MHC complex that can augment the TCR/MHC-peptide interaction. CD8 should therefore be considered as an active participant in the T-cell recognition complex, rather than simply as an accessory molecule.

Antigen presentation and T cell development in H2-M-deficient mice.

Abstract: HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.

Cloning differentially expressed mRNAs

Abstract: Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-γ). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

Biological consequences of thrombin receptor deficiency in mice.

Abstract: The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR -/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR -/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetylcholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR -/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH 2 and TFLLRNPNDK-NH 2 . Our results indicate that ThrR deficiency has a strong impact on fetal development ; however, ThrR -/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

Hla-do is a lysosomal resident which requires association with hla-dm for efficient intracellular transport

Abstract: The murine MHC class II molecule H2-O is expressed in B-cells and in thymic epithelium but the human equivalent, HLA-DO (DO), has not been detected, though the corresponding genes, HLA-DNA and HLA-DOB, are well known. Here we show DO to be a lysosomal resident in B-cells. Surprisingly, DO forms stable complexes with HLA-DM (DM), another lysosomal class II-like molecule which is important for class II-restricted antigen presentation. Association with DM is necessary for efficient exit of DO from the endoplasmic reticulum (ER) and thus for accumulation in lysosomes. The association is evolutionarily conserved and in mice lacking H2-M, the mouse equivalent of DM, the amount of intracellular H2-O is decreased and only minor amounts of H2-O appear to leave the ER. The DO-DM complexes survive in the lysosomal system suggesting that DO and DM functions may be intertwined.

Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kb-restricted epitopes.

Abstract: In the present study, a systematic analysis of the influenza (Flu) PR8 determinants recognized by H-2b mice was undertaken. A single Db-restricted immunodominant epitope (NP(366)) was previously known in this system. Twenty-three different Flu PR8-derived peptides that bound either Kb or Db molecules in vitro were identified. Sixteen were immunogenic following peptide immunization of C57BL/6 mice, yet CTL induced by peptide immunization recognized PR8-infected target cells only in the case of the NP(366) and NS2(114) epitopes. Similarly, CTL responses following whole-PR8 virus immunization were detected only for the same two determinants. CTL recognizing these dominant epitopes had high avidity for peptide-pulsed target cells, with 5 to 200 pM of peptide required for 30% specific lysis. In contrast, most (80%) of the remaining epitopes were recognized with lower avidity (30% effective concentration in the range of 0.4-50 nM). Repeated in vitro stimulation of primary CTL cultures revealed one additional Kb-restricted epitope (M1(128)). This peptide bound Kb with high affinity (4.6 nM) and induced CTL that effectively recognized PR8-infected cells. These results suggest that 1) this epitope is produced by natural processing in relatively high amounts and 2) low precursor frequency might be related to the subdominant status of the M1(128) epitope. Taken together, these results illustrate the crucial contributions of MHC-binding capacity, and T cell repertoire availability, to the shaping of the repertoire of CTL specificities for Flu Ag virus.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells

Abstract: Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

Differing roles for B7 and intercellular adhesion molecule-1 in negative selection of thymocytes.

Abstract: To ensure self tolerance, immature thymocytes with high binding affinity for self peptides linked to major histocompatibility complex (MHC) molecules are eliminated in situ via apoptosis (negative selection). The roles of two costimulatory molecules, B7-1 and intercellular adhesion molecule-1 (ICAM-1), in negative selection was examined by studying apoptosis of T cell receptor transgenic CD4+8+ thymocytes cultured with specific peptides presented by MHC class I-transfected Drosophila cells. When coexpressed on these cells, B7-1 and ICAM-1 act synergistically and cause strong class 1-restricted negative selection of thymocytes. When expressed separately, however, B7-1 and ICAM-1 display opposite functions: negative selection is augmented by B7-1, but is inhibited by ICAM-1. It is notable that B7-1 is expressed selectively in the thymic medulla, whereas ICAM-1 is expressed throughout the thymus. Because of this distribution, the differing functions of B7-1 and ICAM-1 may dictate the sites of positive and negative selection. Thus, in the cortex, the presence of ICAM-1, but not B7-1, on the cortical epithelium may preclude or reduce negative selection and thereby promote positive selection. Conversely, the combined expression of B7-1 and ICAM-1 may define the medulla as the principal site of negative selection.

Induction of heat-stable antigen expression by phagocytosis is involved in in vitro activation of unprimed CTL by macrophages.

Abstract: Costimulation is required for activation of unprimed CTL. While the costimulatory molecules B7 and heat stable Ag (HSA) play a role in CTL response induction generated by dendritic cells, HSA but not B7 contributes to primary CTL response induced by macrophages (M phi), but only if particulate material has been ingested. This is a finding that correlates well with the observation that soon after phagocytosis of latex beads by M phi, cell surface expression of HSA rapidly increases. This increase could not be prevented by addition of drugs that blocked the synthesis or intracellular transport of newly synthesized HSA. However, inhibitors of protein kinase C did significantly down-regulate HSA expression. Other proteins appear to be regulated by a similar mechanism, because the surface expression of the CD45 isoform B220, of IL-2R, and of CD26 also increased immediately following ingestion of beads by M phi. These data suggest that there exists a sequestered pool of proteins that can be exposed coordinately at the cell surface via a protein kinase signaling mechanism that detects phagocytic events. In the case of HSA, we suggest that the ability to rapidly modulate the cell surface level of costimulatory molecules is a useful mechanism by which M phi are able to quickly up-regulate their T cell stimulatory capabilities during the time when, most likely, they are presenting foreign Ag.

Genomic organization of a mouse MHC class II region including the H2-M and Lmp2 loci.

Abstract: The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3′ exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.

Molecular mechanisms of class I major histocompatibility complex antigen processing and presentation.

Abstract: The presentation of antigenic peptides by class I major histocompatibility complex molecules plays a central role in the cellular immune response, since immune surveillance for detection of viral infections or malignant transformations is achieved by CD8+ T lymphocytes which inspect peptides, derived from intracellular proteins, bind to class I molecules on the surface of most cells. The transporter associated with antigen processing selectively translocates cytoplasmically derived peptides of appropriate sequence and length into the lumen of the endoplasmic reticulum where they associate with newly synthesized class I molecules. The translocated peptides are generated by multicatalytic and multisubunit proteasomes which degrade cytoplasmic proteins in a ATP-ubiquitin-dependent manner. This review discusses our current molecular understanding of class I antigen processing and presentation.

Antigen‐presenting Function of the Mouse CD1 Moleculea

Abstract: CD1 molecules are distantly related to major histocompatibility complex (MHC)-encoded class I molecules, and they are coexpressed with beta2 microglobulin (beta2m). In the mouse, CD1 is expressed by intestinal epithelial cells and also by some cells in spleen and lymph node. We have shown that surface expression of mouse CD1 (mCD1) is not dependent upon a functional transporter associated with antigen processing (TAP). This, and other data, suggest that mCD1 may acquire peptides in an intracellular compartment other than the endoplasmic reticulum, where classical class I molecules bind peptide. mCD1 molecules also are distinct from classical class I molecules with regard to the types of peptides that they bind. We have demonstrated that mCD1 molecules preferentially bind peptides much longer than the 8-9 amino acids typical of the peptides that bind to classical class I molecules. The sequence motif for mCD1 peptide binding is characterized by the presence of bulky and hydrophobic amino acid side chains. We have generated mCD1-restricted and peptide-specific T-cell lines, thereby demonstrating the immunologic relevance of peptide binding to mCD1. The reactive T cells are TCR alphabeta+ and CD8+, a phenotype typical of many lymphocytes in both lymph node and intestinal mucosae. We speculate that mCD1 molecules may be capable of sampling peptides from the gut lumen and presenting them to mucosal T lymphocytes. In this way, they may function in the maintenance of normal mucosal homeostasis, and perhaps also in the induction of systemic tolerance to antigens delivered by the oral route. In summary, CD1 molecules are a novel category of antigen-presenting molecules that have features in common with class I molecules, features in common with class II, and properties distinct from either subset of antigen-presenting molecules. Further studies of the antigen-presenting function of these molecules are certain to yield new insight into immune regulation and perhaps also into the mechanism of oral tolerance.

The Thymus and T Cell Death

Abstract: Through the combined effects of positive and negative selection, T cell differentiation in the thymus generates a repertoire of mature T cells that is tailored to tolerate self antigens but mount strong responses to foreign antigens1,2. Thymic selection is associated with extensive cell death, and only a very small proportion of thymocytes are selected for survival and export to the extrathymic environment. This article provides an overview of thymic selection and the fate of thymocytes.

Antigen-specific activation of unprimed cd8+ t cells

Abstract: An artificial antigen-presenting system provides methods and compositions for the activation of unprimed CD8+ T cells in vitro to form specific activated CD8+ T cells (cytotoxic T lymphocytes, or CTL). CD8+ T cells are activated by contacting them with macrophages that present a MHC molecule complexed to an immunogenic peptide, acquired by phagocytosis of the peptide on an artificial support and with an assisting molecule.

Principles of Antigen Processing and Presentation

Abstract: Publisher Summary Getting rid of the host of invading pathogens is a formidable task considering the wide variety of pathogenic organisms and their ability to rapidly mutate when put under evolutionary selection. T cells, which have a central role in regulating immune responses, have antigen-specific receptors with extensive primary sequence homology to immunoglobulins of B cells, but despite this homology the receptors of B and T cells recognize antigens in different ways. While the B cell receptor recognizes structural epitopes in proteins, T-cell receptors recognize antigens in the form of peptides associated with class I or class II molecules encoded in the major histocompatibility complex (MHC). Antigen processing is the process of peptide generation from antigenic proteins and the association of these peptides with MHC molecules. It is of crucial importance for T cell function and thus for the function of the immune system. The differences between the class I and class II antigen-presenting molecules are a reflection of the intracellular trafficking of these molecules and the compartments where they encounter peptides. While both class I and class II molecules assemble in the endoplasmic reticulum (ER) only class I molecules acquire peptides in this compartment. The peptides loaded onto class I are generated in the cytoplasm and transported into the ER. In addition to the normal class I and class II molecules, this chapter describes a number of class I-like molecules. It summarizes the present knowledge of the cell biology of MHC class I and class II antigen processing and presentation, as well as the current understanding of the biology and function of nonclassical class I molecules.

Lymphocyte—Epithelial Cross-Talk in the Intestine: Do Nonclassical Class I Molecules Have a Big Part in the Dialogue?

Abstract: Publisher Summary This chapter focuses on the behavior and properties of nonclassical class I molecules expressed by mouse intestinal epithelial cells, and the possible role these molecules may have in the development and function of mouse intestinal intraepithelial lymphocytes (IEL). IEL have a phenotype that is distinct from other T-cell populations, and these lymphocytes may be the product of a separate, thymus-independent lineage. If such a lineage exists, it would be likely that the intestinal epithelium plays a major role in its development. It is also possible, however, that some IEL are derived from thymus-derived T cells. This is evidenced by the ability of conventional CD4+ cells to home to the gut epithelium in scid mice, and to acquire characteristics of the T cells that normally reside in the intestine. This chapter shows that nonpolymorphic class I molecules expressed in the intestine have unique properties, most notably, their lack of a requirement for TAP in order to be expressed on the cell surface. This lack of a TAP requirement may provide some insight into the specialized function of the TL antigen and mCDl in the mucosal immune system. This chapter further demonstrates that mCDl can function as an antigen-presenting molecule, and that it presents peptide antigens distinct from those presented by classical class I molecules.