The effect of the proteasome inhibitor lactacystin on the presentation of transporter associated with antigen processing (TAP)-dependent and TAP-independent peptide epitopes by class I molecules

Cells were treated with two proteolytic inhibitors, N-acetyl-leucyl-leucyl-norleucinal and lactacystin, the latter reported to be a specific inhibitor for the proteasome. Both inhibitors retarded the maturation of endo-H-resistant forms of murine and human class I molecules from their endo-H-sensitive precursors in cell lines with functional TAP proteins. HLA-A2 maturation readily occurs in TAP-deficient T2 cells, and it has been shown that the peptides associated with A2 are derived from the leader segment of proteins in the secretory pathway. This maturation is inhibited by N-acetyl-leucyl-leucyl-norleucinal but not lactacystin, indicating that the proteasome is not required for the generation of HLA-A2 binding peptides in these cells. The murine class Ib molecule Qa-1b presents a leader peptide derived from D-end class I molecules to alloreactive CTL. Since this presentation is dependent on the expression of TAP proteins, we determined if this requirement reflects a need for the proteasome to process this peptide. We found that lactacystin did not inhibit the maturation of endo-H-resistant forms of Qa-1b that are dependent on this leader peptide for its maturation, nor did it inhibit the expression of this peptide-Qa-1b complex in a functional assay. Thus, unlike conventional cytosolic peptides, leader peptides (regardless of whether they are dependent on TAP for their presentation) do not require the proteasome for processing.     

Molecular analysis of presentation by HLA-A2.1 of a promiscuously binding V3 loop peptide from the HIV-envelope protein to human cytotoxic T lymphocytes

P18(IIIB) is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18(IIIB) for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18(IIIB)-specific CTL line was generated from and HLA-A2(+), HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18(IIIB) seen by murine CTL restricted by H-2Dd. A panel of Ala-substituted peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCR-binding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine I10-specific CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.     

Presentation of a T cell receptor antagonist peptide by immunoglobulins ablates activation of T cells by a synthetic peptide or proteins requiring endocytic processing

T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell-mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (Ig-PLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR-specific T cells may be the operative mechanism for Ig-PLP-LR-mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell-mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.     

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.     

Inhibition of angiotensin converting enzyme and potentiation of bradykinin by retro-inverso analogues of short peptides and sequences related to angiotensin I and bradykinin

There is pharmacological evidence indicating that, in addition to the inhibition of angiotensin converting enzyme (ACE; EC 3.4.15.1), the potentiation of bradykinin (BK) responses may also involve the BK receptor or some binding site in the structures involved in the contractile response to this peptide. Dipeptides such as Val-Trp and some of its analogues as well as tripeptide homologues, including total and partial retro-inverso peptides, were synthesized and assayed for their ability to inhibit purified guinea pig plasma ACE and to potentiate the action of BK on the isolated ileum of the same species. The peptides containing the P2-P1, P1-P'1, and P'1-P'2 inverted amide bonds inhibited ACE, were resistant to hydrolysis, and, depending on the amino acid composition, some of them potentiated the contractile response to BK while others did not. Des-[Arg1]-BK, which has an intrinsic activity at concentrations higher than 10(-5) M, and the very dissimilar angiotensin I (AI) analogue [Cys5-Cys10]-angiotensin-I-(5-10)-amide, which has no detectable contractile activity, were able to inhibit ACE and potentiate BK. In contrast to these peptides, BPP5a and BPP9a from Bothrops jararaca venom, and Potentiators B and C from Agkistrodon halys blomhoffi venom were more effective as BK potentiators than as ACE inhibitors. In conclusion, we have synthesized and assayed compounds that preferentially inhibit ACE, e.g. retro-inverso tripeptides, or potentiate the response of smooth muscle to BK, e.g. snake venom peptides.     

The anti-MUC1 monoclonal antibody BCP8 can be used to isolate and identify putative major histocompatibility complex class I associated amino acid sequences

MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5-17.5 min. The protocol used for the MUC1+ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 molecules by the HLA.A2.1-specific MAb, MA2.1, and high-performance liquid chromatography fractionation of the acid-eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at approximately 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.     

Major histocompatibility complex and T cell receptor interaction of the P91A tum- peptide

The P91A antigen was identified following mutation of P1 mastocytoma cells. The peptide epitope is encoded by a mutant form of the S3 subunit of the PA700 proteasome regulatory complex. P91A stimulates a strong CD8+ T cell response when expressed on tumor cells or normal tissue and P91A-specific T cells express a restricted range of T cell receptors. Although it is a strong Ld-binding peptide, P91A does not conform to the established motif for this major histocompatibility complex (MHC) molecule and this has hampered elucidation of the precise epitope. Ld predominantly associates with nonamer peptides; however, using a variety of complementary approaches, the P91A epitope is identified as the octamer QNHRALDL. In the absence of the Ld motif residue proline at position 2, residues 5-7 are primarily involved in MHC interaction. P91A is thus atypical in its interaction with Ld. Residues 1, 3, and 4 are found to influence T cell recognition of P91A. Definition of the P91A peptide will allow studies on P91A processing and interactions of the P91A peptide/MHC complex with T cell receptors of differing avidity to establish the basis for restricted T cell receptor usage. The basis for the failure of the P91A tum+ peptide (QNRRALDL) to bind to Ld is addressed by molecular modeling.     

Inhibition of experimental autoimmune myasthenia gravis by major histocompatibility complex class II competitor peptides results not only in a suppressed but also in an altered immune response

To assess the capacity of major histocompatibility complex (MHC) class II-binding competitor peptides in inhibiting antibody-mediated disease processes, we studied experimental autoimmune myasthenia gravis in Lewis rats. Experimental autoimmune myasthenia gravis, a disease model mediated by T cell-dependent autoantibodies against acetylcholine receptors, was induced by immunization with Torpedo californica acetylcholine receptor emulsified in complete Freund's adjuvant. The immunodominant acetylcholine receptor T cell epitope was recognized by T cells in the context of MHC class II RT1.B(L). The disease inhibitory capacity of RT1.B(L)-binding peptides not related to the acetylcholine receptor was determined upon co-immunization with Torpedo acetylcholine receptor. Co-immunization of peptide OVA323-339, a strong RT1.B(L)-binding competitor peptide, resulted in complete disease inhibition. Although, the priming of the anti-acetylcholine receptor T cell response was not fully inhibited, the kinetics of the response was changed. Moreover, besides a drastic reduction of the anti-Torpedo acetylcholine receptor antibody titers, a shift in isotype distribution was found. These findings indicate that antibody-mediated autoimmune processes can be suppressed by MHC class II competitor peptides. Furthermore, the administration of such peptides in vivo not only passively inhibits T cell activation, but also functionally alters the immune response.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Intracellular transport of class I MHC molecules in antigen processing mutant cell lines

Intracellular transport and stability of class I MHC glycoproteins depends on the assembly of H chain, beta 2-microglobulin, and peptide. The Ag processing mutant cell lines T2 and RMA-S have defects in peptide loading of class I, resulting in reduced cell surface expression of class I molecules. Expression of class I molecules in the murine cell line RMA-S can be induced at 26 degrees C, suggesting that they are transported to the cell surface, but are unstable. However, most human class I molecules in T2 are poorly expressed at the cell surface, even at 26 degrees C. To directly compare the transport of human and mouse alleles in RMA-S and T2, the human alleles HLA-A2, A3, and B27 were transfected into RMA-S along with human beta 2-microglobulin, and the mouse alleles H-2Kb and Db were transfected into T2. Surface expression of HLA-A3 and B27 in RMA-S remained less than 10% of wild-type levels at 26 degrees C. H-2Kb and Db in both cell lines, however, were expressed at 20 to 30% wild-type levels at 37 degrees C and could be induced to wild-type levels at 26 degrees C or with peptides. The selective expression of murine class I glycoproteins at the cell surface of T2 is not because of their greater stability when associated with human beta 2m, since H-2Kb and Db H chain/human beta 2m complexes dissociate more rapidly in vitro than HLA-A3 and B27 complexes. These results suggest that the difference in transport between human and mouse class I in T2 reflects a fundamental structural property of the class I glycoproteins.     

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.     

Interaction of pigeon cytochrome c-(43-58) peptide analogs with either T cell antigen receptor or I-Ab molecule

We determined that a pigeon cytochrome c-derived peptide, p43-58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43-58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43-58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vbeta usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR beta chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR beta chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.     

Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis

To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.     

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.     

Interaction of murine MHC class I molecules with tapasin and TAP enhances peptide loading and involves the heavy chain alpha3 domain

In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Dendritic cells, interleukin 12, and CD4+ lymphocytes in the initiation of class I-restricted reactivity to a tumor/self peptide

Cell-mediated immunity involving CD8+ lymphocytes is effective in mediating rejection of murine mastocytoma cells bearing P815AB, a tumor-associated and self antigen showing similarity to tumor-specific shared antigens in humans. Although this antigen may act as an efficient target for class I-restricted responses in immunized mice, neither P815AB expressed on tumor cells nor a related synthetic nonapeptide will activate unprimed CD8+ cells for in vivo reactivity, measured by skin test assay. We review evidence showing that the failure of P815AB to initiate CD8+ cell reactivity may be due to defective recruitment of accessory and Th1-like cells to the afferent phase of the response initiated by transfer of mice with dendritic cells pulsed in vitro with the P815AB peptide. Although the copresence of a T helper peptide in dendritic cell priming in vitro with P815AB may compensate for the poor generation of accessory and Th1 cells in the adoptively transferred mice, recombinant IL-12 can replace the helper peptide in both effects. Effective priming to P815AB in vivo is achieved by either exposing dendritic cells to IL-12 prior to P815AB priming or administering the recombinant cytokine in vivo. Different approaches suggest that IL-12 may act both on accessory cells to improve presentation of previously undescribed class II-restricted epitopes of P815AB and on CD4+ cells to improve recognition of such epitopes. In particular, at the CD4+ cell level, IL-12 apparently acts as an adjuvant and an inhibitor of anergy induction. These data offer useful information for developing vaccination strategies using dendritic cells and class I-restricted tumor peptides in humans.     

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to H-2Aq and H-2Ap molecules

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.     

Characterization of peptides that bind the tumor-associated Thomsen-Friedenreich antigen selected from bacteriophage display libraries

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.