A point mutation in the human transporter associated with antigen processing (TAP2) alters the peptide transport specificity

The heterodimeric transporter associated with antigen processing (TAP1/TAP2) translocates peptides from the cytosol into the endoplasmic reticulum where loading of major histocompatibility complex class I molecules takes place. TAP transporters from different species are known to exhibit distinct transport specificities with regard to the C-terminal amino acid (aa) of peptides. Thus, human TAP (hTAP), and rat TAP (rTAP) containing the rTAP2^a allele are rather promiscuous, whereas mouse TAP (mTAP), and rTAP containing the rTAP2^u allele are restrictive and select against peptides with C-terminal small polar/hydrophobic or positively charged aa. The structural basis for this selectivity is not clear. To assess the relative contribution of the TAP1 and TAP2 subunits to transport specificity, we have constructed and analyzed interspecies TAP hybrids and point mutants of hTAP2 expressed in Sf9 insect cells and in TAP-deficient T2 cells. Transport assays with 20 C-terminal variants of the peptide RYWA-NATRSX showed that: first, transport specificity with regard to C-terminal aa is mainly influenced by TAP2, but TAP1 can also contribute. Second, the selective transport of peptides with C-terminal positively charged aa is critically controlled by the amino-terminal region (1–361) on the TAP2 chain, while transport of peptides with C-terminal small polar/hydrophobic aa is determined by residues located within as well as outside the region 1–361. Third, a single point mutation in hTAP2 (374A → D) resulted in a drastic alteration of the transport pattern. These results indicate that both TAP1 and TAP2 contribute to efficient peptide transport and that single point mutations in hTAP2 are able to alter the peptide transport specificity. This opens the possibility that naturally occurring mutations in one of the hTAP subunits may alter epitope selection in vivo.     

Analysis of the fine specificity of rat,mouse and human TAP peptide transporters

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.     

Translocation of long peptides by transporters associated with antigen processing (TAP)

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8–11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2^a, rat TAP1/2^u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8–12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2^a showed a less stringent length selection than rat TAP1/2^u and human TAP. The superior transport of the decamer of the TNKT . Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (K_D = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10⁵ peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.     

Epitope selection in major histocompatibility complex class I-mediated pathway is affected by the intracellular localization of an antigen

We analyzed the mode of antigen presentation of an endogenous antigen localized in the cytoplasm or in the mitochondria. Pseudomonas aeruginosa PAO leucine-, isoleucine-, valine-binding protein (LIVAT-BP) encoded by the braC gene was used as a model antigen. Using mouse BALB/3T3 cells, we established two LIVAT-BP transfectants by transfection of a plasmid harboring the intact braC or braC gene fused with the mitochondrial transport signal derived from the yeast COXIV gene. One of the resulting transfectants, BC-15, expressed LIVAT-BP in the cytoplasm, while YZ-710 cells expressed LIVAT-BP in the mitochondria. The splenic effector cells derived from BALB/c mice primed with BC-15 cells exhibited cytotoxic T lymphocyte (CTL) activity against BC-15 cells, but not against YZ-710 cells, whereas splenic effector cells primed with YZ-710 cells exhibited CTL activity against YZ-710 cells, but not against BC-15 cells. Neither group of splenic effector cells showed CTL activity against parental BALB/3T3 cells. These CTL belonged to the CD8⁺ αβ T cell subset. Furthermore, we observed that the CTL activity against BC-15 cells or YZ-710 cells was blocked with anti-H2-K^d mAb, but not with anti-H2-D^d or H2-L^d mAb. The CTL against BC-15 or YZ-710 cells could kill parental BALB/3T3 cells in the presence of peptides produced by alkali lysis of the LIVAT-BP. suggesting that these CTL indeed recognized the peptide(s) derived from LIVAT-BP. We determined that the epitope for the CTL against BC-15 cells was QYGEGIATEV, corresponding to residues 162–171, and that the epitope recognized by the CTL against YZ-710 cells was GYKLIFRTI, corresponding to residues 123–131 of LIVAT-BP, respectively. Thus, we show here that epitope selection for MHC class I expression is affected by the intracellular localization of the antigenic protein.     

Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP-independent presentation

Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1^−/− mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1^−/− or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257–264 epitope from ovalbumin [OVA(257–264)], which binds the mouse class I molecule K^b. The source of the OVA(257–264) epitope was either the Crl-OVA(257–264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1^−/− mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257–264) for recognition by OVA(257–264)/K^b-specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1^−/− macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257–264) was presented 100 times less efficiently when the source of OVA(257–264) was full-length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1^−/− macrophages, which also depended upon the source of the OVA (257–264) epitope (Crl-OVA versus full-length OVA). These data suggest that TAP1^−/− and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257–264) epitope influences the extent of TAP-independent processing for MHC class I presentation.     

The effect of anchor residue modifications on the stability of major histocompatibility complex class I-peptide interactions

Anchor residues in peptides determine the specificity of binding to major histocompatibility complex class I molecules through interactions of their side chains with pockets in the peptide-binding groove. We have compared the kinetics of association of a Sendai virus nucleoprotein-derived peptide (FAPGNYPAL, termed SV9) with H-2K^b class I molecules, and the same peptide iodinated on the anchor residue tyrosine (¹²⁵I-SV9). Even though the association rates were too rapid for direct measurements, competition studies indicated that they were similar for SV9 and ¹²⁵I-SV9. To measure the binding of non-radioactive SV9 directly, SV9 was tritiated (³H-SV9). ³H-SV9 remained stably associated with H-2K^b molecules, whereas ¹²⁵I-SV9 dissociated in a temperature-dependent fashion. Thus, modifications on anchor residues do not necessarilly have to affect the specificity and association kinetics of peptide binding to class I molecules but can affect the stability of the resulting class I-peptide interaction. The dissociation of peptides with modified and, more generally, suboptimal anchor residue side chains may explain the presence of empty class I molecules and free class I heavy chains at the cell surface.     

Stress protein (hsp73)-mediated,TAP-independent processing of endogenous,truncated SV 40 large T antigen for Db-restricted peptide presentation

Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the con-stitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two D^b-binding peptides and one K^b-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two D^b-binding epitopes, but not the K^b-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi D^b molecules is available for 2–3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.     

Expression of TAP1 by human trophoblast

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Antitransporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G⁺ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.     

TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity

Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8⁺ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8⁺ T cell population in TAP1 -/- mice. CD8⁺ T cells were polyclonal with regard to T cell receptor (TCR) Vβ expression. Overall, Vβ usage in TAP1 -/- mice appeared to be very similar to that in wild-type mice, with significantly reduced levels of Vβ5.1/5.2-expressing CD8⁺ T cells as the only clear exception. This polyclonal population of CD8⁺ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2^b class I. The present results demonstrate that a polyclonal CD8⁺ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8⁺ T cells.     

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.     

The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza-specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome-dependent degradation plays an important role in the cytosolic generation of CTL epitopes.     

TAP-dependent major histocompatibility complex class I presentation of soluble proteins using listeriolysin

Immunization of mice with mixtures of listeriolysin, a pore-forming hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, together with soluble ovalbumin, nucleoprotein of influenza virus, or β-galactosidase of Escherichia coli, resulted in strong cytotoxic CD8 T cell responses to each of the respective passenger proteins in vivo. Also, the concomitant addition of either protein with listeriolysin to target cells elicited efficient sensitization of these cells which could be attributed to the pore-forming activity of listeriolysin. This response was dependent upon a functional TAP transporter and was inhibitable by brefeldin A, indicating the transfer of the soluble proteins into the cytosol and the classical major histocompatibility (MHC) class I presentation pathway. The treatment of target cells with listeriolysin under our experimental conditions did not affect cell viability and the pores generated by listeriolysin treatment were repaired within 60 min. Introduction of soluble proteins into the MHC class I presentation pathway by listeriolysin provides a powerful system to study the cytotoxic response towards intracellular pathogens and would allow for rapid screening of potential antigens in vaccine formulations.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

TAP2-defective RMA-S cells present Sendai virus antigen to cytotoxic T lymphocytes

The murine antigen-processing-defective mutant cell line RMA-S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)-defective RMA-S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA-S cells required 2-3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA-S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild-type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA-S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings comfirmed leakiness of antigen presentation in RMA-S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I-mediated antigen presentation.     

A soluble,single-chain Kd molecule produced by yeast selects a peptide repertoire indistinguishable from that of cell-surface-associated Kd

Peptide binding to a soluble, single-chain K^d protein produced by the yeast strain Kluyveromyces lactis, and to K^d molecules on K^d-expressing cells (P815) was studied using radiolabeled K^d-restricted peptides. The stability of the peptide-K^d complexes formed was monitored in the absence and presence of unlabeled competitor peptides. Radioiodination of the Tyr anchor residue in position 2 of the peptide interferes with binding. A K^d-biased peptide library and a modified antigenic peptide in which a second Tyr was added in positions 6 and 8, respectively, were therefore used to assay binding. Recombinant and cell-associated K^d molecules are very similar in the following respects: the ease with which the proteins can be loaded with labeled peptide; the spectrum of peptides selected from a peptide library; the stability of the labeled peptide-K^d complex formed; and the ability to partially dissociate the class 1-peptide complex with exogenous, unlabeled peptides. These results imply that measurements of peptide binding to soluble K^d molecules are a reliable indicator of the peptide-binding properties of K^d proteins on living cells. The large quantities of soluble recombinant K^d protein currently available represent an invaluable tool not only for dissecting the molecular mechanisms of antigen presentation but also for vaccinations and the design of T cell-specific toxins.     

Real-time measurement of antigenic peptide binding to empty and preloaded single-chain major histocompatibility complex class I molecules

Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single-chain K^d (SC-K^d) molecule in which the K^d heavy chain was connected by a 15-residue link to β₂-microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated K^d-restricted peptides to SC-K^d resulted in significant fluorescence enhancement, which could be inhibited by unmodified K^d-restricted peptides. Real-time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263–260 peptide bound to “empty” SC-K^d with an association rate constant of 1140 M^?1s^?1, and the subsequent spontaneous dissociation of the SC-K^d-peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253–260 peptide, but with a slower association constant for unlabeled peptide, 77 M^?1s^?1. Thus, the K^d-peptide complex on the surface of antigen-presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on K^d before export to the surface. The apparent activation energy for PbCS 253–260 peptide binding to SC-K^d was 6.78 ± 0.64 kcal/mole, similar to values previously reported for antigen-antibody interactions.     

The murine gamma-herpesvirus-68 MK3 protein causes TAP degradation independent of MHC class I heavy chain degradation

The murine gamma-herpesvirus-68 MK3 protein has an intricate interaction with the peptide loading complex that involves MK3 stabilization, a rapid degradation of MHC class I heavy chains, and a slower degradation of TAP. Here we have used tapasin chimeras to distinguish functionally the different immune evasion mechanisms of MK3. Tapasin was cloned in two alternatively spliced forms that differed by a single transmembrane valine residue. Each restored antigen presentation and MK3 function in tapasin-deficient cells. The transmembrane/cytoplasmic portion of tapasin, linked to the extracellular domain of CD8, also restored TAP stability and MK3 stability in tapasin-deficient cells. MK3 did not associate with or degrade MHC class I in these cells, which lacked the endoplasmic reticulum domain of tapasin, but degraded TAP at least as efficiently as when full-length tapasin was present. The un-degraded MHC class I consequently showed impaired maturation. The fact that MK3 required intact tapasin to degrade MHC class I but only the transmembrane/cytoplasmic portion of tapasin to degrade TAP indicated that these two immune evasion functions operate independently.     

Major histocompatibility complex class I molecules interact with both subunits of the transporter associated with antigen processing,TAP1 and TAP2

Prior to loading antigenic peptides, assembled major histocompatibility complex (MHC) class I molecules associate with the transporter associated with antigen processing (TAP) in a complex which also includes calreticulin and a recently described component, tapasin. The interaction of MHC class I molecules has been characterized as occurring exclusively with the TAP1 chain of the TAP heterodimer. In contrast, as described here, in the TAP-deficient human cell line T2, MHC class I molecules interact with a transfected rat TAP2 polypeptide in addition to rat TAP1. Furthermore, this interaction with TAP2 also involves calreticulin and tapasin. An association with both TAP polypeptides would presumably further enhance the efficiency of peptide loading of MHC class I molecules by allowing more than one MHC class I allele proximity to the site of peptide supply on each TAP complex.     

Heat shock increases antigenic peptide generation but decreases antigen presentation

The heat shock response is a universal and highly conserved cellular response to stress. We describe here the effect of elevated temperature on the capacity of B cells to present antigen. Heat shock markedly affects the ability of these cells to process and present tetanus toxin to class II-restricted T cell clones. Inhibition of antigen presentation is due neither to a modification of antigen capture nor to a variation of major histocompatibility complex (MHC) class II molecule synthesis and cell surface expression. Stressed and nonstressed B cells are able to present peptides loaded at the cell surface with the same efficiency. Nevertheless, heat shock leads to an increase of antigen peptide generation in subcellular compartments; an enhancement of cathepsin B activity is also observed. These data suggest that such a stress induces a failure in the intracellular peptide loading onto MHC class II molecules.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.