Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma.

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8(+) T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-specific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer(+) CD8(+) T cells allowed the isolation of tetramer(bright) and tetramer(dull) populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1(157-165)) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

A simple and rapid procedure for the purification of synthetic polypeptides by a combination of affinity chromatography and methionine chemistry.

Chemical synthesis of bioactive peptides has become a widespread and rapidly growing technique due to automated and efficient protocols for chain assembly. For most applications, the crude synthetic product must be purified to remove residual reactants, failure sequences and chemically modified peptide species. We propose here a method of universal applicability based on immobilized metal ion affinity chromatography, CNBr cleavage and use of reversible Met-sulfoxide protection. With this method we were able to purify to homogeneity in high yield the PbCS 242-310 polypeptide corresponding to the C-terminal region of Plasmodium berghei CS protein.

The diversity of antigen-specific TCR repertoires reflects the relative complexity of epitopes recognized.

Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 Kd. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252-260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170-179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant. H-2 Kd molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/Kd complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.

Synthesis of a radioiodinated photoreactive MAGE-1 peptide derivative and photoaffinity labeling of cell-associated human leukocyte antigen-A1 molecules.

The synthesis of a photoreactive derivative of the human leukocyte antigen-A1 (HLA-A1)-restricted MAGE-1 peptide 161-169 (EADPTGHSY) is described. Using conventional automated solid-phase peptide synthesis, a photoreactive derivative of this peptide was synthesized by replacing histidine-167 with photo-reactive N-beta-4-azidosalicyloyl-L-2,3-diaminopropionic acid. The C-terminal tyrosine was incorporated as phosphotyrosine. This peptide derivative was radioiodinated in the presence of chloramine T. This iodination took place selectively at the photoreactive group, because the phosphate ester prevented tyrosine iodination. Following dephosphorylation with alkaline phosphatase and chromatographic purification, the radiolabeled peptide derivative was incubated with cells expressing HLA-A1 or other HLA molecules. Photoactivation resulted in efficient photoaffinity labeling of HLA-A1. Other HLA molecules or other cellular components were not detectably labeled. This labeling was inhibited by HLA-A1 but not by HLA-A2-binding peptides. This synthesis is generally applicable and can also be adapted to the synthesis of well-defined radiolabeled nonphotoreactive peptide derivatives.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

MHC class I H-2Kd-restricted antigenic peptides: additional constraints for the binding motif.

The previously defined binding motif of MHC class I H-2Kd-restricted antigenic peptides consists of a Y residue in position P2 and a hydrophobic residue with a large aliphatic side chain (L, I, or V) in position P9/P10 of optimal 9- or 10-mer peptides. We show now that the presence of a charged or a F residue in position P5 reduces the Kd-restricted competitor activity of several cytotoxic T lymphocyte (CTL) epitopes and model peptides, at a degree comparable to A substitutions for the P2 or the P9/P10 anchor residues. Various charged, polar, aromatic, and aliphatic amino acids were substituted for S256 in the CTL epitope Plasmodium berghei circumsporozoite (CS) 253-260 8-mer and in its CS 252-260 9-mer form, whereas a more restricted panel of substitutions was tested in the CTL epitopes influenza nucleoprotein 147-155 9-mer and HLA-CW3 170-179 10-mer. Analysis of all the Kd-restricted epitopes so far defined also revealed an uncharged residue at this position. These additional structural constraints present in the Kd binding motif may thus improve the prediction of new epitopes recognized by T cells in the context of this MHC molecule.

Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design.

The design, total chemical synthesis, and immunological properties of a four-alpha-helix bundle template-assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58-74 of the alpha 1 heavy chain domain of HLA-A2 was modeled in order to increase helix stability and amphiphilicity of the 17-mer peptide, preserving the residues for potential T-cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four-helix-bundle topology of the assembled peptide blocks. After extensive HPLC purification, stepwise solid-phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial alpha-helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four-alpha-helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA-A2-positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.

Studies on the interaction of ribonuclease inhibitor with pancreatic ribonuclease involving differential labeling of cysteinyl residues.

Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.

N-glycosylation of HIV-gp120 may constrain recognition by T lymphocytes.

The HIV envelope protein gp120 is heavily glycosylated, having 55% of its molecular mass contributed by N-linked carbohydrates. We investigated the role of N-glycosylation in presentation of HIV-gp120 to T cells. T cell clones obtained from humans immunized with a recombinant nonglycosylated form of HIV-gp120 (env 2-3) were studied for their ability to recognize both env 2-3 and glycosylated gp120. We found that 20% of CD4+ T cell clones specific for env 2-3 fail to respond to glycosylated gp120 of the same HIV isolate. Using synthetic peptides, we mapped one of the epitopes recognized by such clones to the sequence 292-300 (NESVAINCT), which contains two asparagines that are glycosylated in the native gp120. These findings suggest that N-linked carbohydrates within an epitope can function as hindering structures that limit Ag recognition by T lymphocytes.

C3 synthetic peptides support growth of human CR2-positive lymphoblastoid B cells.

The nature of CR type 2 (CR2)-ligand interactions which leads to the activation of human B cells was analyzed by using synthetic peptides and CR2-positive cell lines. The third component of C (C3) supported the growth of human lymphoblastoid B cells in serum-free medium containing human transferrin. This effect was inhibited by an antibody to C3d (mAb 130) which specifically inhibits C3d binding to CR2, but not by other anti-C3 mAb. Synthetic peptides corresponding to the CR2-binding site on C3d, P28 (residues 1187-1214) or multivalent P13 [1202-1214)4-template), supported the proliferative response of CR2-positive human lymphoblastoid lines in a similar way as C3 and this response could be inhibited by the anti-CR2 mAb OKB7. The proliferative response to C3 or peptides was dose dependent and a 60-fold higher concentration of P28 peptide was required to induce the same level of proliferation as C3. This stimulation of growth was observed only on CR2 expressing cell lines Raji and Daudi, and not on the CR2-negative Burkitt lymphoma cell line Rael and the monocytic cell line U937. In contrast to the stimulatory effect of P28 and P13-template, monomeric P14 (1201-1214) was not able to support the growth of these cell lines. This peptide, however, inhibited the proliferative response of the CR2-positive lines to C3, P28, and multivalent-P13, thus indicating that cross-linking of the CR2 receptor is necessary for B cell proliferation. Another peptide, E12 (from glycoprotein (GP)350, the major EBV outer membrane GP) which shows a high degree of similarity with P14, also inhibited the proliferative response of Raji cells, suggesting that this segment on GP350 is involved in the interaction of EBV with CR2. The possibility of using the above peptides as well as other peptides with "tailor-made" structure in studying the multifunctional role of C3 is discussed.

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis.

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.

Cell surface proteins reacting with activated complement components.

The biologic activities mediated by the products of complement activation include cellular, bacterial, and viral lysis, inflammation, phagocytosis, and immunoregulation. These responses are achieved through the interaction of the activated forms of several of the complement proteins with cell membrane proteins. This report reviews aspects of the structure, ligand specificity, and function of the various complement receptors with particular emphasis on those receptors which bind to the activated fragments of C3. In addition, we briefly summarize the surface proteins on foreign particles that bind C3 and their possible role in the pathogenesis of these organisms.

Two adjacent epitopes on a synthetic dodecapeptide induce lactate dehydrogenase B-specific helper and suppressor T cells.

The outcome of an immune response to the enzyme lactate dehydrogenase B (LDH-B) is determined by the interplay between two types of regulatory T lymphocytes, T helper (Th) and T suppressor (Ts) cells. Most mouse strains are capable of generating Th but not Ts cells, and are therefore high responders to LDH-B in terms of both antibody production and antigen-specific T-cell proliferation. However, in strains expressing the b or k allele at the E beta locus of the major histocompatibility complex (Mhc), Ts cells are induced that partly or totally abrogate the proliferative response of Th cells to LDH-B. As a result, these strains are phenotypically medium (E beta b expressors) or low (E beta k expressors) responders. Because the suppression in the LDH-B system is antigen-specific (i.e. it only affects LDH-B-specific Th cells), it is conceivable that the Th and Ts cells use the antigen itself to communicate with each other. To investigate this possibility, we set out to determine which epitopes of the LDH-B molecule are recognized by Th and Ts cells. On the basis of previous studies, a loop structure extending from residue 211 to residue 224 of pig LDH-B appeared to be preferentially recognized by most Th-type (class II Mhc-restricted, proliferating) clones. By using a synthetic peptide, we demonstrate here that both Th and Ts cells are induced by the 211-222 stretch of LDH-B sequence. The use of two further dodecapeptides, each with a single amino-acid substitution in comparison with the pig 211-222 sequence, has revealed that Th and Ts cells have different fine specificities. Thus the loop appears to have two closely linked, if not overlapping, epitopes, one recognized by Th and the other by Ts cells. This finding is consistent with two possible mechanisms of suppression, namely bridging of Th and Ts cells by antigen and subsequent transmission of a suppressive signal, and competition for antigen between Th and Ts cells.

Fine specificity analysis of lactate dehydrogenase B-specific proliferating T cell clones: implications for the mechanism of alloreactivity.

T cell clones of C57BL/6 origin which recognize porcine lactate dehydrogenase B (LDH-BP) together with the Ab molecule were characterized in terms of fine specificity for both LDH-B and self-major histocompatibility complex determinants. Using antigen-presenting cells from the Ab-mutant strain B6.C-H-2bm12 (bm12), three clonotypes could be distinguished: the first responds to LDH-BP + bm12, the second fails to respond and the third is alloreactive to bm12. The last clone exhibits additional alloreactivities to A molecules expressed in strains of H-2 haplotypes f, r, s, u, w6, w7, w16, w17 and w23. All three clonotypes give identical response patterns to a panel of 17 different dehydrogenase enzymes, and react to the same tryptic peptide of LDH-BP. Thus, these clones appear to recognize the same LDH-B epitope together with at least 3 different determinants of the Ab molecule. The data suggest that alloreactivity is more closely related to T cell specificity for self-major histocompatibility complex than to specificity for foreign antigen.