HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences

Previous studies showed that HLA-E was expressed in lymphoblastoid cell line (LCL) 721.221 cells, but surface expression was lacking. To determine the signals controlling surface expression, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner, a hybrid of HLA-E was identified, designated AEH, which differed from HLA-E by having the HLA-A2 signal sequence substituting for the HLA-E leader peptide. Transfection of LCL 721.221 cells with AEH induced HLA-E surface expression. Analysis of peptides bound to HLA-E revealed that a nonamer peptide derived from the A2 signal sequence was the predominant peptide bound. LCL 721.221 cells transfected with certain class I genes, including HLA-G, were also sufficient to promote peptide binding and HLA-E surface expression without increasing the level of HLA-E heavy chain synthesis. Peptides bound to HLA-E consisted of nine amino acids, with methionine at position 2 and leucine in the carboxyl-terminal position, and were nearly identical to the leader sequence-derived peptide previously shown to be a predominant peptide bound to the murine Qa-1 Ag. Signal peptides derived from certain HLA-B proteins with threonine in position 2 only marginally up-regulated HLA-E surface expression in .221 cells. An examination of HLA-E peptide binding in the TAP negative cell line .134 indicated that peptide binding to HLA-E was dependent on a functional TAP heterodimer regardless of whether peptide was available in cis, as in the AEH construct, or in trans, as in the class I transfectants of .221 cells.     

Different sources of acidity in glucose-elicited extracellular acidification in the yeast Saccharomyces cerevisiae

Qa-1b binds a peptide (AMAPRTLLL), referred to as Qdm (for Qa-1 determinant modifier), derived from the signal sequence of murine class Ia molecules. This peptide binds with high affinity and accounts for almost all of the peptides associated with this molecule. Human histocompatibility leukocyte antigen (HLA)-E, a homologue of Qa-1b, binds similar peptides derived from human class Ia molecules and interacts with CD94/NKG2 receptors on natural killer cells. We used surface plasmon resonance to determine the ability of Qa-1b to bind related ligands representing peptides derived from the leaders of class I molecules from several mammalian species. All of the peptides reported to bind HLA-E bound readily to Qa-1b. In addition, peptides derived from leader segments of different mammals also bound to Qa-1b, indicating a conservation of this "Qdm-like" epitope throughout mammalian evolution. We have attempted to define a minimal peptide on a polyglycine backbone that binds Qa-1b. Our previous findings showed that P2 and P9 are important but not sufficient for binding to Qa-1b. Although a minimum peptide (GMGGGGLLL) bound Qa-1(b), its interaction was relatively weak, as were peptides sharing five or six residues with Qdm, indicating that multiple native residues are required for a strong interaction. This finding is consistent with the observation that this molecule preferentially binds this single ligand.     

The proteasome inhibitor lactacystin prevents the generation of an endoplasmic reticulum leader-derived T cell epitope

The presentation of viral antigens on MHC class I molecules requires their intracellular fragmentation into peptides of appropriate length and anchor residue positions. Evidence has accumulated that the proteasome is the endoprotease in charge of the generation of MHC class I ligands in the cytoplasm. The generation of T cell epitopes derived from the leader peptides of endoplasmic reticulum (ER) targeted proteins, however. has been reported to be independent of the proteasome. Here we show that the H-2Db restricted antigen presentation of the immunodominant T cell epitope derived from the ER leader of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) is completely abolished by administration of the proteasome inhibitor lactacystin. Thus our data support the role of the proteasome in class I restricted antigen processing and extend it to an ER leader derived epitope from a viral glycoprotein.     

TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide

Under most circumstances, cell surface MHC class I molecules display peptides derived from a cytosolic pool of proteins. The efficient presentation of such peptides requires the functioning of two MHC gene products [TAP1 and TAP2 (transporter-associated with Ag processing 1 and 2)] that form a complex that facilitates transmembrane movement of peptides from the cytosol to the endoplasmic reticulum, the site of peptide association with class I molecules. It has been previously shown that peptides can be presented in a TAP-independent manner in association with HLA A2.1 or H-2 Kd if they are expressed COOH-terminal to an endoplasmic reticulum insertion/signal sequence derived from the adenovirus E3/19K glycoprotein (Anderson et al., 1991. J. Exp. Med. 174: 489; Eisenlohr et al., 1992. Cell 71: 963). We show that: 1) the E3/19K signal sequence greatly enhances the presentation of each of four additional peptides tested in association with H-2 Kb or Kk, 2) the E3/19K signal sequence can be substituted by a signal sequence derived from beta-IFN, and 3) the E3/19K signal sequence does not function when located at the COOH terminus of antigenic peptides. These findings indicate that first, many peptides require TAP for efficient presentation to T cells, second, expression of peptides COOH-terminal to signal sequences is a generally applicable method of bypassing the TAP-dependence of peptide presentation and third, the leader sequence does not act to bypass TAP simply by increasing the hydrophobic nature of peptides.     

Maturation of Qa-1b class I molecules requires beta 2-microglobulin but is TAP independent

Two conformationally distinct and stable forms of Qa-1b, one strongly associated with beta 2-microglobulin (beta 2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable beta 2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without beta 2m, there was no maturation of either form in cells from beta 2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires beta 2m for an initial folding step. However, beta 2m is not essential for subsequent processing of Qa-1b molecules.     

Analysis of posterior plagiocephaly: deformational versus synostotic

Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcgammaR), which mediate internalization of antigen-IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II-restricted antigen presentation. We now show that FcgammaRs have two additional specific attributes in murine DCs: the induction of DC maturation and the promotion of efficient MHC class I-restricted presentation of peptides from exogenous, IgG-complexed antigens. Both FcgammaR functions require the FcgammaR-associated gamma chain. FcgammaR-mediated MHC class I-restricted antigen presentation is extremely sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules, ICs should efficiently sensitize DCs for priming of both CD4(+) helper and CD8(+) cytotoxic T lymphocytes in vivo.     

HLA-A*0201 presents TAP-dependent peptide epitopes to cytotoxic T lymphocytes in the absence of tapasin

Tapasin is a 48-kDa endoplasmic reticulum (ER)-resident glycoprotein that binds to the transporter associated with antigen processing (TAP) and mediates an interaction between TAP and newly synthesized MHC class I molecules. It is also essential for the proper antigen presenting function of HLA-A*0101 (HLA-A1), HLA-A*0801 (HLA-B8) and HLA-B*4402 (HLA-B4402). We show here that while tapasin is required for HLA-A*0201 (HLA-A2) molecules to bind to TAP, its absence does not block the presentation of HLA-A2-restricted TAP-dependent epitopes to cytotoxic T lymphocytes indicating that, unlike HLA-A1, HLA-B8 and HLA-B4402, HLA-A2 has access to the TAP-dependent peptide pool even in the absence of tapasin. Nevertheless, the overall efficiency with which HLA-A2 was loaded with optimal, stabilizing peptides was impaired in the cell line .220, resulting in a significant increase in the fraction of HLA-A2 molecules being released from the ER in a "peptide-receptive" state.     

A novel antagonist, phenylbenzene omega-phosphono-alpha-amino acid, for strychnine-sensitive glycine receptors in the rat spinal cord

Leukaemia-specific proteins may be recognized by T-lymphocytes as neoantigens if peptides corresponding to mutated sequences bind to major histocompatibility complex (MHC) molecules on leukaemic cells. We studied the ability of a series of synthetic peptides corresponding to the junctional sequences of BCR/ABL proteins to bind to class I molecules in two human cell lines, LBL 721.174 (T2) (HLA-A2, B5) and BM36.1 (HLA-A1, B35), and one murine cell line RMA-S (H-2Kb, Db). These cell lines are defective in intracellular peptide loading of class I molecules, resulting in markedly reduced cell surface class I expression: class I expression can be rescued by provision of peptides binding to the alleles expressed by the mutant cell. Eighteen peptides spanning the junctional sequences of the b2a2 and b3a2 proteins were tested for their ability to rescue expression of the class I alleles borne by these cells using flow cytometry. Allele-specific control peptides known to bind HLA-A2, HLA-B35, H-2Kb and H-2Db increased expression of these alleles 2- to 3-fold: 0/18 BCR/ABL peptides enhanced HLA-A2, HLA-B35 or H-2Kb expression, but three b2a2 peptides consistently increased H-2Db expression. These results suggest that BCR/ABL junctional peptides are unlikely to be presented to T-cells in association with HLA-A2, HLA-B35 or H-2Kb. Conversely, the finding that some b2a2 peptides bind specifically to H-2Db suggests that a murine model of graft-versus-leukaemia (GVL) could be constructed.     

Major histocompatibility complex class I viral antigen processing in the secretory pathway defined by the trans-Golgi network protease furin

Classical antigen presentation by major histocompatibility complex class I molecules involves cytosolic processing of endogenously synthesized antigens by proteasomes and translocation of processed peptides into the endoplasmic reticulum (ER) by transporters associated with antigen presentation (TAP). Alternative pathways for processing of endogenous antigens, generally involving the ER, have been suggested but not fully proved. We analyzed the potential for class I presentation of proteolytic maturation of secretory antigens in the exocytic pathway. We found that hepatitis B (HB) virus secretory core protein HBe can efficiently deliver COOH-terminally located antigenic peptides for endogenous class I loading in the absence of TAP. Antigen presentation to specific cytotoxic T lymphocytes correlates with protein maturation at the COOH terminus, since modification of maturation and transport of HBe through the secretory pathway alters antigen presentation. Both maturation and a necessary processing step occur in the Golgi or post-Golgi compartment. Antigen presentation is independent of proteasome activity, but inhibitors of the trans-Golgi network resident protease furin inhibit both HBe maturation and antigen presentation. These results define a new antigen processing pathway located in the secretory route, with a central role for proteolytic maturation mediated by the subtilisin protease family member furin as an efficient source for antigen presentation.     

Dissociation of proteasomal degradation of biosynthesized viral proteins from generation of MHC class I-associated antigenic peptides

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

TAP1-independent loading of class I molecules by exogenous viral proteins

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1-/-) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glyco- and nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1-/- cells as by control cells. Peptide regurgitation was not involved. Since particulate, non-replicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.     

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

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.     

MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific CTLs

Subclinical infection of BALB/c mice with the intracellular pathogen Listeria monocytogenes results in the development of MHC class Ia- and Ib-restricted CTLs. L. monocytogenes-infected TAP-/- bone marrow macrophage targets are not lysed by MHC class Ia- or Ib-restricted CTLs, showing a requirement for transport of peptides into the endoplasmic reticulum for development of the MHC class Ib-peptide target. L. monocytogenes-infected B6.Tla(a)-derived bone marrow macrophages (Kb Qa-1a) are not lysed by BALB/c (Kd Qa-1b)-derived antilisterial CTLs, confirming an earlier finding that the Ib-restricting element is T region encoded. We have further determined that Qa-1b is a restricting element for antilisterial CTLs using L. monocytogenes-infected Qa-1b-transformed mouse L cells as well as human-derived HeLa cells as target populations. These L. monocytogenes-infected Qa-1b-transformed cell lines are lysed by BALB/c (Qa-1b)- or C57BL/6 (Qa-1b)-derived antilisterial CTLs, but are not lysed by B6.AKM (Qa-1a)-derived antilisterial CTLs. Using L. monocytogenes-infected targets, we found that MHC class Ia- and Ib-restricted CTLs are evident within 4 days following infection, peak on day 5 following infection, and although Ib-restricted CTLs disappear by day 6 postinfection, la-restricted antilisterial CTL activity can still be detected. These results demonstrate that Qa-1b is a restricting element for antilisterial CTLs, and expression of the MHC class Ib-presented target at the cell surface is TAP dependent. In addition, these results show that following L. monocytogenes infection, MHC class Ib-restricted CTLs are evident in vivo.     

A topographic study on the distribution of cisplatin in xenografted tumors on nude mice

The influence of the TAP complex on T-cell allorecognition of MHC class II molecules was examined using human B-cell lines that have mutations in the TAP 1 or 2 genes. The TAP mutations led to the loss of allorecognition for two of 28 anti- HLA-DR T-cell clones. Restoration of TAP expression by transfection of a TAP 2 cDNA clone led to recovery of the alloresponse for both clones. These results could be explained in two ways. First, TAP dependence could reflect specificity for a peptide derived from an MHC class I molecule that is less efficiently generated by the endocytic pathway in the TAP-deficient stimulator cells owing to reduction in surface class I expression. The proliferative responses of these clones to the TAP-deficient stimulator cells was not restored by rescue of cell-surface expression of class I molecules by low temperature culture or by the addition of class I-binding peptides. These data therefore favor the alternative explanation that class II loading by some peptides is TAP dependent. Circumstances that lead to the amplification of this minority pathway of endogenous presentation by class II MHC molecules may have the potential to interrupt self-tolerance.     

Reappraisal of bicarbonate secretion by the human oesophagus

TAP1 and TAP2 molecules are involved in the transport of peptides prior to their association with class I molecules and are mandatory for efficient antigen presentation. To investigate whether loss of expression of TAP1 or TAP2 is a likely mechanism of immune escape in malignant melanoma, TAP1 and TAP2 mRNA was analyzed by RT-PCR in 39 melanoma cell lines expressing at least 2 of the known melanoma-associated antigens, tyrosinase, Melan-A/MART-1, gp100, MAGE-1 and MAGE-3. All 39 cell lines expressed both TAP1 and TAP2 at the mRNA level. To investigate other factors potentially involved in immune escape, the expression of LMP2, LMP7, HLA class I molecules, beta2-microglobulin (beta2m) and specific HLA-A alleles was evaluated by RT-PCR and FACS analyses. All 39 cell lines expressed LMP2, LMP7 and beta2m. A single cell line (FM37) had lost the expression of class I molecules, and this same cell line showed loss of expression of the HLA-A2 heavy chain. No cell lines showed loss of expression of the HLA-A1 heavy chain. Based on our studies of in vitro established cell lines, loss of TAP1/2 or LMP2/7 expression does not appear to be a common mechanism of immune escape in malignant melanoma.     

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.     

Linkage of LMP, TAP, and RING3 with Mhc class I rather than class II genes in the zebrafish

The LMP2 and LMP7 genes code for subunits of the proteasome, a multimeric enzymatic complex that degrades proteins into peptides. The two subunits replace corresponding constitutively expressed subunits during the immune response. Some of the peptides generated by the proteasome in the cytosol are transported by the products of the TAP1 and TAP2 genes into the lumen of the endoplasmic reticulum and are loaded onto the assembling MHC class I molecules. In mammals, the LMP2, LMP7, TAP1, and TAP2 genes reside in the class II region of the Mhc, closely linked to the RING3 gene. In the present study we identified, cloned, and sequenced the LMP, TAP2, and RING3 genes of the zebrafish, Danio rerio. We identified variants of these genes and used them in a segregation analysis of haploid embryos derived from heterozygous mothers. The analysis revealed that in zebrafish, the LMP2, LMP7, TAP12, and RING3 loci are closely linked but, in contrast to mammals, the LMP/TAP/RING3 cluster resides not in the Mhc class II but in the class I region. We also confirmed that in the zebrafish, the class I and class II regions are not linked to each other. In this species, therefore, the LMP/TAP/RING3 genes are clustered with the class I genes on a chromosome that apparently does not contain any class II genes. The linkage of LMP/TAP/RING3/class I may be the original and the LMP/TAP/RING3/class II a derived arrangement of these genes.     

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.     

MHC class I molecules compete in the endoplasmic reticulum for access to transporter associated with antigen processing

We have used the functionally distinct TAP alleles of the rat in cellular transfectants as tools to investigate how newly formed rat class I (RT1.A) molecules with distinct peptide requirements gain access to suitable peptides in the endoplasmic reticulum (ER). Normal maturation of RT1.Aa depends on the presence in the ER of peptides with C-terminal arginine, while restrictive TAP-B allelic group transporters fail to transport such peptides. In this situation, RT1.Aa is retained in the ER. We show that this retention is accompanied by accumulation of RT1.Aa in the ER, partly associated with TAP and partly free. In such cells, access to TAP of a second allelic product, RT1.Au, which does not require C-terminal arginine peptides, is competitively inhibited by the build-up of RT1.Aa. Nevertheless, RT1.Au loads and matures normally. Introduction of a permissive TAP-A allele competent to transport C-terminal arginine peptides releases RT1.Aa from the ER and restores RT1.Au interaction with TAP. Both class I alleles associate indiscriminately with permissive and restrictive TAP alleles. The data support the view that interaction with TAP is not a prerequisite for peptide loading by class I molecules, so long as suitable peptides are available in the ER. They further show that TAP association of a class I molecule depends on a competitive balance in the ER defined by the extent to which the peptide requirements of other class I molecules present are satisfied and not only by the intrinsic strength of the interaction with TAP.