Background. 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--producing CD8 super(+) T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. Methods. We employed caged major histocompatibility complex molecules to generate image 250 H-2L super(d) tetramers and to distinguish T. gondii-specific CD8 super(+) T cells in BALB/c mice. Results. We identified 2 T. gondii-specific H-2L super(d)- restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L super(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L super(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. Our results shed light on the identity and the parasite stage-specificity of 2 CD8 super(+) T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.
We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)‐ATPases required ...for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide‐loaded, SDS‐stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B‐treated cells, a slow increase (> 20‐fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface‐disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor‐treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans‐Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.
MHC class II molecules associate, during biosynthesis, with peptides derived from endocytosed antigen. Here, Jacques Neefjes and Hidde Ploegh describe the intracellular transport of MHC class II ...molecules and its relationship to the binding of peptides in endosomal compartments. They discuss alternative routes for the delivery of antigen to sites at which peptides associate with MHC class II molecules and raise the possibility of cell type-specific differences in the handling of MHC class II molecules, and hence in antigen presentation.
Human CMV (HCMV) infection leads to an almost complete inhibition of expression of MHC class I proteins. After infection with HCMV, the biosynthesis of HLA class I molecules was examined in human ...lung fibroblasts and in mouse fibroblasts transfected with genes encoding the human class I H chain HLA-B27 and human beta 2-microglobulin (beta 2m). In both cell types, we observed a large decrease in steady state levels specific for human class I H chains--both free H chains and those complexed with beta 2m. In the mouse cells transfected with HLA class I, infection did not affect levels and assembly of mouse class I H chains with human beta 2m. The effect of HCMV infection on class I expression is insensitive to phosphonoacetic acid, suggesting the involvement of immediate early or early viral proteins. Pulse-chase analysis showed that the low steady state level of class I H chains in HCMV-infected cells is not the result of a reduced rate of synthesis. Rather, we observed accelerated degradation of class I H chains, regardless of their association with beta 2m. We conclude that HCMV reduces human MHC class I protein levels by interference with the stability of class I H chains.
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 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.
The transporter associated with antigen processing (TAP) delivers peptides to the lumen of the endoplasmic reticulum in an adenosine triphosphate (ATP) dependent fashion for presentation by major ...histocompatibility complex class I molecules. We show that the mouse TAP translocator (H-2b haplotype) selects peptides based on a minimal size of nine residues, and on the presence of a hydrophobic COOH-terminal amino acid. The preponderance of COOH-terminal hydrophobic amino acids in peptides capable of binding to mouse class I molecules thus fits remarkably well with the specificity of the TAP translocator. In addition to transport in the lumenal direction, efflux of peptide in the cytosolic direction is observed in an ATP- and temperature-dependent manner. By maintaining a low peptide concentration at the site of class I assembly, this efflux mechanism may ensure that class I molecules are loaded preferentially with high affinity peptides.
The herpes simplex virus (HSV) immediate early protein ICP47 inhibits the transporter associated with antigen processing (TAP)-dependent peptide translocation. As a consequence, empty major ...histocompatibility complex (MHC) class I molecules are retained in the endoplasmic reticulum and recognition of HSV-infected cells by cytotoxic T lymphocytes is abolished. We chemically synthesized full-length ICP47 (sICP47) and show that sICP47 inhibits TAP-dependent peptide translocation in human cells. Its biological activity is indistinguishable from that of recombinant ICP47 (rICP47). By using synthetic peptides, we mapped the core sequence of ICP47 minimally required for TAP inhibition to residues 2-35. This segment is located within the region of the molecule conserved between ICP47 from HSV-1 and HSV-2. Through alanine scanning substitution we identified three segments within this region that are critical for the ability to inhibit TAP function. The interaction of ICP47 with TAP is unlikely to mimic precisely that of the transported peptides, as deduced from differential labeling of the TAP1 and TAP2 subunits using sICP47 fragments with chemical cross-linkers.
The human high affinity receptor for IgE (FcɛRI) is a cell surface structure critical for the pathology of allergic reactions. Human FcɛRI is expressed as a tetramer (αβγ
2
) on basophils or mast ...cells and as trimeric (αγ
2
) complex on antigen-presenting cells. Expression of the human α subunit can be down-regulated by a splice variant of FcɛRIβ (β
var
). We demonstrate that FcɛRIα is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to αβγ
2
and αγ
2
, we demonstrate the presence of αβ and αβ
var
γ
2
complexes that are stable in the detergent Brij 96. The role of individual FcɛRI subunits for the formation of functional, immunoglobulin E–binding FcɛRI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: β and γ support ER insertion, signal peptide cleavage and proper N-glycosylation of α, whereas β
var
allows accumulation of α protein backbone. We show that assembly of FcɛRI in the ER is a key step for the regulation of surface expression of FcɛRI. The ER quality control system thus regulates the quantity of functional FcɛRI, which in turn controls onset and persistence of allergic reactions.
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