Search PubMed⌕ Search

Biomedical subjects

Peter Cresswell

Publications and source records attributed to Peter Cresswell.

31 records · Page 2Linked to original sources
PubMed2004

Recent developments in MHC-class-I-mediated antigen presentation.

Defective ribosomal products provide an important supply of endogenous peptides for entry into the classic MHC class I antigen presentation pathway. The recruitment of endoplasmic reticulum membrane during phagosome biogenesis allows exogenous antigens to be translocated into the cytosol as well as providing access to the class I peptide transport and loading machinery. This combination of features provides a mechanism for cross-presentation by specialised antigen presenting cells. Recent studies have shed new light on these pathways and have also described the emerging K3 family of viral ubiquitin E3 ligases, which constitutively ubiquitinate and degrade MHC class I molecules and other immunoreceptors.

Animals↗
PubMed2004

Cellular mechanisms governing cross-presentation of exogenous antigens.

The recent discovery of fusion of endoplasmic reticulum membrane with nascent phagosomes suggests that this peripheral compartment in macrophages and dendritic cells may serve as an organelle optimized for major histocompatibility complex (MHC) class I-restricted cross-presentation of exogenous antigens. The process allows intersection of the endosomal system with the endoplasmic reticulum, the classical site of MHC class I peptide loading, and may reconcile the seemingly conflicting evidence indicating both of these sites are crucial in cross-presentation. Here we discuss the potential mechanisms involved in loading exogenous antigens onto MHC class I molecules and the implications of this new evidence for the in vivo function of dendritic cells.

Animals↗
PubMed2003

Tapasin is a facilitator, not an editor, of class I MHC peptide binding.

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.

Antigen Presentation↗
PubMed2003

Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.

Conventionally, MHC class I-restricted antigen (Ag) processing requires the action of the multimolecular peptide-loading complex within the endoplasmic reticulum (ER). Here we show that early phagosomes from human dendritic cells (DCs) contain the peptide-loading complex, incorporating MHC class I, beta2 microglobulin, transporter associated with Ag processing (TAP), calreticulin, tapasin, and ERp57. Antigenic peptides could be translocated into purified phagosomes by TAP and loaded onto cognate class I molecules, inducing their specific dissociation from the loading complex. Endoglycosidase H-sensitive class I molecules were detected at the DC cell surface, suggesting that these molecules traffic there directly from phagosomes. Macropinocytosis also allowed internalized soluble Ags access to an ER-like compartment containing the class I loading complex. Blockade of TAP by endocytosis of a soluble derivative of human cytomegalovirus protein US6 confirmed that, although retrotranslocation into the cytosol is critical for processing, efficient association of class I molecules with peptides derived from exogenous Ags occurs within a compartment directly accessible to internalized proteins. Together, this evidence suggests that early phagosomes and pinosomes facilitate cross presentation of exogenous Ags by DCs.

Antigen Presentation↗
PubMed2003

Regulation of MHC class I transport in human dendritic cells and the dendritic-like cell line KG-1.

Dendritic cells (DCs) progress through distinct maturational phases; immature DCs capture Ag while mature DCs are optimized for Ag presentation. Proper control of immunity requires regulated compartmentalization of MHC class II molecules. We report that DCs also regulate MHC class I trafficking throughout maturation. Although mature human DCs express high levels of surface MHC class I, immature DCs exhibit lower surface levels while retaining MHC class I-peptide complexes in the Golgi. A cell line, KG-1, behaves similarly. We confirm the similarity of KG-1 to DCs by demonstrating its capacity to present exogenous Ags in an MHC class I-restricted fashion to CD8(+) T cell hybridomas, a phenomenon called cross-presentation. Biochemical characterization of MHC class I trafficking throughout maturation showed that, in early KG-1 dendritic-like cells, surface arrival of MHC class I-peptide complexes is delayed by their retention in the Golgi. In mature dendritic-like cells, these complexes relocate to the surface and their stability increases, concomitant with up-regulation of costimulatory molecules. Maturation induces qualitative changes in the MHC class I-associated peptide repertoire demonstrated by increased thermostability. The differential processing of MHC class I throughout maturation may prevent premature immune activation while promoting T cell responses in lymph nodes to Ags acquired at sites of inflammation.

Animals↗
PubMed2002

Calnexin, calreticulin, and ERp57 cooperate in disulfide bond formation in human CD1d heavy chain.

Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and beta(2)-microglobulin (beta(2)m) in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need beta(2)m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with beta(2)m. These differences led us to investigate early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. Here we show that CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin.

1-Deoxynojirimycin↗
PubMed2002

Identification of specific glycoforms of major histocompatibility complex class I heavy chains suggests that class I peptide loading is an adaptation of the quality control pathway involving calreticulin and ERp57.

Glycosylation analysis was used to probe the sequence of events accompanying the binding of antigenic peptides to the major histocompatibility complex class I heavy chains. Free heavy chains were isolated from the beta(2)-microglobulin-negative cell line Daudi and from the B-lymphoblastoid cell line Raji. Heavy chains were also isolated from Raji cells in multimolecular complexes (peptide loading complexes) containing the transporter associated with antigen processing, tapasin and ERp57 with and without the lectin-like folding chaperone, calreticulin. Calreticulin is a soluble protein that recognizes primarily the terminal glucose of Glc(1)Man(7-9)GlcNAc(2) glycans. This paper shows that monoglucosylated glycoforms of heavy chain, which exist transiently in the endoplasmic reticulum in the initial stages of the glycosylation processing pathway, are present in the peptide loading complex. The data are consistent with a model in which the release of peptide-loaded major histocompatibility complex class I molecules from calreticulin, induced by deglucosylation of the heavy chain N-linked glycan, signals the dissociation of the complex. This is consistent with the hypothesis that the class I loading process is an adaptation of the quality control mechanism involving calreticulin and ERp57.

Calreticulin↗
PubMed2002

Role of the C-terminal propeptide in the activity and maturation of gamma -interferon-inducible lysosomal thiol reductase (GILT).

gamma-Interferon-inducible lysosomal thiol reductase (GILT) is constitutively expressed in antigen-presenting cells. GILT facilitates unfolding of endocytosed antigens in MHC class II-containing compartments by enzymatically reducing disulfide bonds. The enzyme is synthesized as a 35-kDa precursor. Although a fraction of the precursor is secreted as a disulfide-linked dimer, the majority is directed via the mannose-6-phosphate receptor pathway to endocytic compartments where its N- and C-terminal propeptides are cleaved to generate the 30-kDa mature form. Both precursor and mature GILT reduce disulfide bonds with an acidic pH optimum. In this report, we show that the cysteine residues in the C-terminal propeptide, Cys-211 and Cys-222, serve key structural roles. Mutation of Cys-222 abolishes disulfide-linked dimerization of precursor GILT and decreases the efficiency of GILT maturation. Mutation of Cys-211 results in both impaired intracellular maturation and loss of enzymatic activity of the precursor form at an acidic pH. A similar phenotype was obtained upon mutation of Cys-200, which is retained in the mature form. Cys-200 and Cys-211 seem to form a disulfide bond that links the propeptide and the mature enzyme until reduction in the lysosome. This disulfide bridge is essential for stability of the enzyme at low pH and for its proper maturation in vivo.

Amino Acid Sequence↗
PubMed2002

Absence of gamma-interferon-inducible lysosomal thiol reductase in melanomas disrupts T cell recognition of select immunodominant epitopes.

Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of gamma-interferon-inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILT's ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses.

Antigen-Presenting Cells↗
PubMed2002

Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules.

CD1 family members are antigen-presenting molecules capable of presenting bacterial or synthetic glycolipids to T cells. Here we show that a subset of human CD1d molecules are associated with major histocompatibility complex (MHC) class II molecules, both on the cell surface and in the late endosomal/lysosomal compartments where class II molecules transiently accumulate during transport. The interaction is initiated in the endoplasmic reticulum with class II-invariant chain complexes and appears to be maintained throughout the class II trafficking pathway. A truncated form of CD1d which lacks its cytoplasmic YXXZ internalization motif is transported to late endosomal/lysosomal compartments in the presence of class II molecules. Furthermore, the same CD1d deletion mutant is targeted to lysosomal compartments in HeLa cells expressing class II molecules and invariant chain by transfection. The deletion mutant was also found in lysosomal compartments in HeLa cells expressing only the p33 form of the invariant chain. These data suggest that the intracellular trafficking pathway of CD1d may be altered by class II molecules and invariant chain induced during inflammation.

Animals↗
PubMed2002

Disulfide reduction in major histocompatibility complex class II-restricted antigen processing by interferon-gamma-inducible lysosomal thiol reductase.

Constitutively expressed in antigen-presenting cells (APCs), interferon-gamma-inducible lysosomal thiol reductase (GILT) catalyzes disulfide bond reduction under acidic conditions. GILT contains a CXXC motif similar to the WCGH/PCK motif of proteins in the thioredoxin family. This class of enzymes catalyzes, at a neutral pH, dithiol oxidation, disulfide bond reduction, and disulfide bond isomerization. A well-established assay spectrophotometrically measures interchain disulfide bond reduction of insulin via the precipitation of aggregating free B chains. However, the insolubility of insulin at low pH limits the use of this assay. To assess the thiol reductase activity of GILT, we employed an assay that uses denatured [125I]F(ab')2 as a substrate, which is detailed in this article. In addition, we discuss approaches used to demonstrate the mechanism of action of GILT and to identify substrates.

Animals↗
PubMed2002

Disulfide bond isomerization and the assembly of MHC class I-peptide complexes.

The presence of a disulfide bond inside the peptide binding groove of MHC class I molecules and of the thiol oxidoreductase ERp57 in the class I loading complex suggests that disulfide bond isomerization may play a role in peptide loading. Here we show that ERp57 and tapasin are disulfide linked inside the loading complex. Mutagenesis of cysteine 95 in tapasin not only abolishes formation of the ERp57-tapasin bond but also prevents complete oxidation of the class I heavy chain in the loading complex. The resulting MHC class I-beta2m heterodimers are poorly loaded with high-affinity peptides in the ER but nevertheless escape to the cell surface where they are unstable. These findings suggest a role for disulfide bond isomerization in tapasin-mediated peptide loading.

Antiporters↗