Analysis of the mechanism for extracellular processing in the presentation of human immunodeficiency virus-1 envelope protein-derived peptide to epitope-specific cytotoxic T lymphocytes

An immunodominant epitope of human immunodeficiency virus-1 (HIV-1) gp160 recognized by Dd class I major histocompatibility complex (MHC) molecule-restricted, CD8+ cytotoxic T lymphocytes (CTL) was originally identified as a peptide composed of 15 amino acids (P18IIIB: RIQRGPGRAFVTIGK). However, further study has indicated that a 10-mer peptide, I-10 (RGPGRAFVTI), within P18IIIB is the minimal-sized epitope and the trimming step(s) of two carboxyl terminal amino acids (GK) is essential to produce I-10 from P18IIIB. In the processing, angiotensin-1-converting enzyme (ACE), found in sera, plays a central role in generating I-10. Target cells could be sensitized with I-10 under conditions where ACE activity in the sera was abrogated. In contrast, in the case of P18IIIB, requiring further processing to delete the C-terminus of two amino acids in order to act, sensitization of target cells was completely abrogated under the conditions. Pretreatment of target cells with brefeldin A (BFA), preventing the presentation of endogenous antigens from the class I MHC molecule pathway, did not inhibit the presentation of P18IIIB. Moreover, glutaraldehyde-fixed cells, which can not process native protein, though they could present the exogenously added peptides, were also sensitized by P18IIIB. These results clearly demonstrate that the fine processing to produce I-10 occurred in the extracellular milieu. Furthermore, our result suggests that the longer P18IIIB can bind to the class I molecules on the cell surface, and then be trimmed by ACE while it is bound. The mechanisms behind the extracellular processing outlined in this paper will offer important information for designing peptide-based vaccines to elicit MHC molecule-restricted effectors.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

Crystal structures and KIR3DL1 recognition of three immunodominant viral peptides complexed to HLA-B*2705

We have solved the crystal structures of three HLA-B*2705-peptide complexes with the immunodominant viral peptides: EBV EBNA3C 258-266 (RRIYDLIEL), influenza (flu) nucleoprotein NP383-391 (SRYWAIRTR), and HIV gag 264-273 (KRWIILGLNK). Long-term non-progression during HIV infection has been associated with presentation by HLA-B*2705, and T cell recognition, of the highly immunodominant KRWIILGLNK peptide. The tight hydrogen-bonding network observed between the HLA-B*2705 B-pocket and the peptide P2 arginine guanadinium anchor explains why mutation of this residue during HIV infection results in loss of peptide binding, immune escape and progression to AIDS. Prominent, solvent-exposed structures within these peptides may participate in generating T cell responses to these immunodominant epitopes. In the HLA-B*2705 complex with flu NP383-391, the amino acid side chains of residues 4, 7 and 8 are solvent-exposed whilst in the HIV decamer, the main-chain bulges into the solvent around P7. Thus, HLA-B*2705 presents viral peptides in a range of conformations. Tetrameric complexes of HLA-B*2705 with the HIV and flu but not EBV peptides bound strongly to the killer-Ig-like receptor (KIR)3DL1. Substitution of EBV P8 glutamate to threonine allowed recognition by KIR3DL1. In the HLA-B*2705-EBV structure the P8 glutamate side chain is solvent-exposed and may inhibit KIR3DL1 binding through electrostatic forces.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2L(d) binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.     

A monoclonal antibody recognizes a subset of the H-2Dd mouse major class I antigens

Binding studies and competition experiments have shown that a monoclonal antibody (mAb) named 28-8-6 recognizes only 5 to 10% of the cell surface Dd molecules. The molecules detected by 28-8-6 mAb appear to be genuine H-2Dd antigens on the basis of their MW and isolectric points. In addition, the detectability of the subset of cell surface Dd molecules by 28-8-6 does not depend on their degree of glycosylation nor on the presence of mouse beta-2-microglobulin. Several interpretations are discussed. mAb 28-8-6 might detect a particular conformation or a particular chemical derivatization of otherwise normal H-2Dd molecules. Also, because the epitope recognized by 28-8-6 lies close to the peptide binding site, it is possible that mAb 28-8-6 recognizes a subset of Dd molecules bearing a certain category of self peptides.     

In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up-regulation of major histocompatibility complex class I molecules by short synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC-I). Externally added peptides can sensitize target cells by binding directly to MHC-I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC-I interactions can also be reflected as up-regulation of MHC-I in vitro on certain cells. We have compared the capacity of Db, Kb- and Ld-binding peptides, which are slightly different in length, to up-regulate MHC-I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9-mer Db-binding peptide by adding cystein to the amino terminus and lysine to the amino- or carboxy terminus and studied the effects on MHC-I up-regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen-binding groove of Db. These small modifications of the optimal 9-mer peptide strongly influenced their functions but still there was a correlation between MHC-I up-regulation and CTL responses. Up-regulation of MHC-I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen-presenting cells in vivo.     

Crossrecognition by CD8 T cell receptor αβ cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp60 which share intermediate sequence homology

Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H-2D^b)-restricted CD8 T cell receptor (TcR) α/β cells. A CD8 TcR α/β clone with specificity for the mycobacterial hsp60 peptide_499–508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon-γ (IFN-γ) stimulation. The stress-induced, self peptide could be extracted from IFN-γ-stressed macrophages by acid elution, suggesting that the IFN-γ-induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60-specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9-mer to 11-mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H-2D^b binding were tested in CTL assays. The overlapping murine hsp60 peptides_{162–170/171} were stimulatory at a concentration as low as 10–100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10–100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN-γ-stressed macrophages to the cross-reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.     

Characterization of a helper T cell epitope recognized by mice of a low responder major histocompatibility type.

Most known helper T cell (Th) epitopes studied have naturally been immunodominant epitopes recognized by T cells from animals of high responder major histocompatibility complex (MHC) haplotype. We have previously found that most such immunodominant Th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other, and the corresponding peptide can usually elicit a response to the native protein. However, very few epitopes seen by MHC low responder T cells have been identified. Within the CNBr fragment of residues 1-55 of sperm whale myoglobin (SwMb), a Th epitope is known to exist that stimulates T cells from low responder H-2k mice, but it has not yet been localized to a length of 8-12 residues, the usual length of a Th epitope. To determine whether this low responder epitope would have similar properties, we located it using 10 evenly overlapping 15-residue peptides that span the region. Analysis of this region by the computer program predicted the site covered by two peptides (residues 26-40 and 31-45 which overlap by 10 residues) to be the most likely site for a Th epitope. Of the 10 peptides tested experimentally, only one peptide (residues 26-40) was able to stimulate two low responder Th clones that are specific for the 1-55 region. The peptide was able to prime T cells of low responder B10.BR mice in vivo for in vitro response to the native SwMb as well as to the peptide fragment of residues 1-55. Immunization of low responder mice with SwMb showed that, of the 10 overlapping peptides, the major site of response within the 1-55 region is to the identified peptide. Finally, an extended peptide of residues 24-42 was made to increase the amphipathic score. This extended peptide induced greater proliferation of the clones. Thus, this low responder epitope has properties similar to those of immunodominant epitopes recognized by high responders.     

Identification of minimal CD8+ and CD4+ T cell epitopes in the Plasmodium yoelii hepatocyte erythrocyte protein 17kDa

Immunization of mice with subunit vaccines based on the Plasmodium yoelii 17kDa hepatocyte erythrocyte protein (PyHEP17), orthologue of Plasmodium falciparum exported protein 1 (PfExp1), induces antigen-specific immune responses and protects against sporozoite challenge. To aid in the characterization of candidate subunit vaccines based on this antigen, we have mapped the immunodominant and subdominant CD8+ and CD4+ T cell epitopes on PyHEP17. Using a panel of 29 15-mer synthetic peptides representing the complete sequence of PyHEP17 (amino acids 1-153), and overlapping each other by 10 residues, we identified an immunogenic region between amino acids 61-85. To define the minimal CD4+ and CD8+ T cell epitopes within this region, we synthesized 25 9-mer peptides overlapping each other by one residue. We screened the capacity of the 15-mer and 9-mer peptides to be recognized by splenocytes and lymph node cells from mice immunized with PyHEP17 plasmid DNA or peptides in Freund's adjuvant, as assessed by cytokine secretion, lymphoproliferation, and cytotoxicity. The profile of response to the T cell epitopes varied depending upon the immunization regimen. Antigen-specific T cell responses were detected to three 15-mer peptides (residues 61-75, 66-80 and 71-85) representing two 10-mer epitopes mapping to residues 66-75 (LTKNKKSLRK) and 71-80 (KSLRKINVAL). IFN-gamma responses after DNA immunization predominantly mapped to two overlapping 9-mer peptides (residues 73-81 and 74-82) sharing an eight amino acid overlap (residues 74-81, RKINVALA), whereas CTL responses predominantly mapped to four 9-mer peptides (residues 61-69, 70-78, 76-84, and 84-92). In addition, a subdominant 10-mer CD8+ T cell epitope recognized by peptide immunization but not DNA immunization mapped to residues 31-40 (GKYGSQNVIK). The identification of these epitopes will allow the evaluation of delivery systems for malaria vaccine candidates as well as the delineation of protective immune mechanisms.     

Identification of naturally processed peptides bound to the class I MHC molecule H-2Kd of normal and TAP-deficient cells

This report details the biochemical features of natural peptides selected by the H-2Kd class I MHC molecule. In normal cell lines, the length of the naturally processed peptides ranged from 8 to 18 amino acids, although the majority were 9-mers (16% were longer than nine residues). The binding motif for the 9-mer peptides was dominated by the presence of a tyrosine at P2 and an isoleucine/leucine at the P9 position. The P2 residue contributed most towards binding; and the short peptides bound better and formed longer-lived cell surface complexes than the long peptides, which bound poorly and dissociated rapidly. The longer peptides did not exhibit this strictly defined motif. Trimming the long peptides to their shorter forms did not enhance binding and conversely, extending the 9-mer peptides did not decrease binding. The long peptides were present on the cell-surface bound to H-2Kd (Kd) and were not intermediate products of the class I MHC processing pathway. Finally, in two different TAP-deficient cells the long peptides were the dominant species, which suggested that TAP-independent pathways selected for long peptides by class I MHC molecules.     

Fine Molecular Specificity of Linear and Assembled Antibody Binding Sites in HIV-1 p24

A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11-kDa 104-mer peptide covering the C-terminal residues 270-373 of the p24 gag protein (HIV-1BRU strain). All monoclonal antibodies recognized HIV-1IIIB infected MOLT3 cells by fluorescence and gave positive Western blot signals with viral gag peptides (p55 and/or p24). Oligopeptide binding regions were located with competitive enzyme-linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104-mer region. The antibodies bound to p24 peptide sequences located within the 275-293 and 351-368 regions. One antibody (LH104-B) which reacted with residues 357-362 bound to p55 alone. In contrast, another antibody (LH104-I), which recognized the residues 358-363, i.e. with five out of six residues in common with antibody LH104-B for its epitope region, reacted exclusively with p24. At least two of the antibodies (LH104-C and -A) which bound to p24 alone, apparently recognized conformational epitopes. They gave positive reactions with the regions 288-293/351-356 and 284-289/351-356, respectively. This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins.     

MHC class I molecules, structure and function

MHC class I molecules (MHC-I) are cell surface recognition elements expressed on virtually all somatic cells. These molecules sample peptides generated within the cell and signal the cell's physiological state to effector cells of the immune system, both T lymphocytes and natural killer (NK) cells. In addition, molecules structurally related to MHC-I, collectively known as MHC-Ib, are more specialized and, in some cases, interact with more limited subsets of lymphoid cells. Using the recently determined structure of the classical MHC-I molecule, H-2Dd, as a paradigm for structure and function, we review other MHC-I and MHC-Ib molecules, with an emphasis on how the same basic structural fold is employed by classical MHC-I molecules to bind specific peptides and T cell receptors, and is exploited by the MHC-Ib molecules in more stringent molecular interactions. It is instructive that structurally related molecules have evolved to perform a number of unique and distinct functions in immune and non-immune recognition.     

Redox-regulated peptide transfer from the transporter associated with antigen processing to major histocompatibility complex class I molecules by protein disulfide isomerase

Most antigenic peptides are generated by proteasomes in the cytosol and are transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind with nascent major histocompatibilitiy complex class I molecule (MHC-I). Although the overall process of peptide-MHC-I complex assembly is well studied, the mechanism by which free peptides are delivered from TAP to MHC-I is unknown. In this study, we investigated the possible role of protein disulfide isomerase (PDI) as a peptide carrier between TAP and MHC-I. Analysis of PDI-peptide complexes reconstituted in vitro showed that PDI exhibits some degree of specificity for peptides corresponding to antigenic ligands of various human leukocyte antigen (HLA) alleles. Mutations of either anchor residues of the peptide ligand or the peptide-binding site of PDI inhibited the PDI-peptide interaction. The PDI-peptide interaction increased under reducing conditions, whereas binding of the peptide to PDI decreased under oxidizing conditions. TAP-associated PDI was predominantly present in the reduced form, whereas the MHC-I-associated PDI was present in the oxidized form. Further, upon binding of optimal peptides, PDI was released from TAP and sequentially associated with HLA-A2.1. Our data revealed a redox-regulated chaperone function of PDI in delivering antigenic peptides from TAP to MHC-I.     

Specific and randomly derived immunoactive peptide mimotopes of mycobacterial antigens

The mycobacterial cell surface contains complex nonprotein antigens that are highly immunoactive in nature. However, these antigens are chemically heterogeneous and structurally complex, thereby limiting their applications. To identify their peptide mimotopes, phage-displayed peptide libraries Ph.D.-7 and Ph.D.-12 were panned on either defined template, monoclonal antibody (MAb) CS-35 against lipoarabinomannan (LAM), or a polyclonal rabbit immune serum reactive against whole cells of Mycobacterium bovis BCG. Panning on anti-LAM MAb CS-35 yielded two confirmed mimotopes of LAM, a 7-mer and a 12-mer, whereas panning on polyclonal serum yielded a large repertoire of mimotopes reactive against sera from BCG-immunized rabbits, one of which turned out to have the same sequence as the 7-mer LAM mimotope. The dissociation constant of the interaction between MAb CS-35 and a synthetic peptide corresponding to the 7-mer LAM mimotope was determined to be 7.55 microM. Dot blot assays were performed with peptides corresponding to the two LAM mimotopes to evaluate their diagnostic potential. Both peptides gave discernibly higher signals with a panel of tuberculosis (TB) patient sera than with sera from healthy controls. The peptides were also found to stimulate the release of tumor necrosis factor alpha and interleukin-12 cytokines in the J774A.1 cell line and primary bone marrow-derived macrophages, indicating that they may have immunomodulatory potential. The present study demonstrates that peptide mimotopes of known and unknown mycobacterial antigens could be isolated by using subtractive phage display techniques and that these peptides could have potential applications in areas such as TB diagnostics and immunotherapy.     

Epitope specificity and isoforms of the mycobacterial 19-kilodalton antigen.

The topography and specificity of B- and T-cell stimulatory epitopes from the 19-kDa protein of Mycobacterium tuberculosis were investigated by using overlapping synthetic peptides. Murine antisera identified two cryptic epitopes (residues 11 to 30 and 61 to 80) and one species-specific immunodominant epitope (residues 140 to 159). Immunoglobulins G1 and G2a antibody isotypes varied for the respective peptide immunogens but without relationship to the T-cell cytokine profiles which were characterized by high gamma interferon and low interleukin 5 levels. Antisera to recombinant M. tuberculosis 19-kDa protein (rGST-19) cross-reacted with homologous proteins of similar size from organisms of the Mycobacterium avium-intracellulare complex. Two-dimensional gel electrophoresis revealed differences in the number, relative mobility, and charge of isoforms of the 19-kDa protein, possibly reflecting posttranslational modifications. The immunodominant T-cell epitope from the M. tuberculosis 19-kDa protein (residues 61 to 80) and the corresponding peptide sequence from Mycobacterium avium subsp. intracellulare (residues 64 to 83), differing at five residues, were both recognized in a genetically permissive manner. Peptides 61-80 and 64-83 stimulated cross-reactive responses in BALB/c (H-2d) mice, while in the C57BL/10 (H-2b) strain, responses to peptide 61-80 were species specific. In purified protein derivative-positive healthy individuals, the M. avium subsp. intracellulare peptide stimulated stronger responses than did the M. tuberculosis peptide, whereas patients with active tuberculosis had enhanced in vitro T-cell responses to both peptides.     

Fine-specificity of the anti-CENP-A B-cell autoimmune response

The major targets recognized by anti-centromere autoantibodies are the three centromere-associated proteins (CENPs) A, B, and C, with apparent molecular masses of 19, 80, and 140 kDa, respectively. Previously a major epitope region on the 19-kDa CENP-A antigen was identified by synthesis of a soluble synthetic 15-mer peptide (amino acids 3-17) to be used in enzyme-linked immunosorbent assay and western blot competition assays. However, no systematic experimental scanning for epitope regions on the CENP-A autoantigen has yet been performed. In this study we scanned the complete CENP-A amino acid sequence for epitopes using 19 previously characterized autoimmune-sera. Overlapping peptides 15 amino acids in length and offset by three amino acids were synthesized on activated membranes, covering the whole CENP-A autoantigen. Probing of the membranes with various anti-centromere sera showed that all epitopes are clustered in the N-terminal 45 amino acids. For fine-mapping of this autoreactive region the N-terminus of CENP-A (amino acids 1-45) was scanned again by probing overlapping 15-mer, 12-mer, 10-mer, 8-mer, 7-mer, 6-mer, and 5-mer peptides, all offset by one amino acid, with anti-centromere sera. In this way we localized two epitope core regions within the N-terminal 45 amino acids, one covering amino acids 2-17, recognized by 17 sera, and the other covering amino acids 22-38, recognized by 18 sera. One serum did not react with CENP-A at all. Several sera seem to recognize overlapping individual epitopes within these two epitope core regions. All sera, however, recognize a sequence motif G/A-P-R/S-R-R.     

H-2-associated effects of flanking residues on the recognition of a permissive mycobacterial T-cell epitope.

Previously we have identified an immunodominant, eight-residue, epitope core sequence (TAAGNVNI) from the 19,000 MW protein of Mycobacterium tuberculosis, which is recognized in the context of multiple H-2 I-A molecules. In this study, the role of residues flanking this T-cell epitope core was examined, using a series of 20 mer analogue peptides in which the native flanking residues were progressively replaced with L-alanine. Analogue peptides were tested for their capacity to stimulate a CD4+ 19,000 MW protein-specific T-cell line, revealing that all but one N-terminal flanking residue could be replaced collectively by alanine without significant loss of stimulatory activity. However, clear H-2-associated differences in the requirement for flanking residues were demonstrated with peptide-specific T-cell hybridomas. In particular, H-2d-derived hybridomas were much more stringent in their requirement for flanking residues than were H-2b hybridomas. All polyalanine-substituted peptides bound I-Ab molecules, with affinities similar to the native unsubstituted peptide. In contrast, significantly reduced binding to I-Ad was observed with several analogue peptides, although without a clear relationship to the degree of substitution. Furthermore, in H-2b mice, neither immunogenicity nor cross-reactivity with the native peptide showed a clear inverse relationship with respect to the degree of alanine substitution. The results presented in this paper indicate that flanking residues can influence T-cell specificity and that these effects may be controlled by major histocompatibility complex (MHC) haplotype.     

Localization of the regions on the C-terminal domain of the heavy chain of botulinum toxin a recognized by t lymphocytes and by antibodies after immunization of mice with pentavalent toxoid

We have mapped the regions recognized by T and/or 6 cells (Abs) on the C-terminal domain (Hc) of the heavy chain of botulinum neurotoxin serotype A (BoNT/A) after immunization of two inbred mouse strains with pentavalent toxoid (BoNTs A, B, C, D and E). Using a set of synthetic overlapping peptides, encompassing the entire Hc domain (residues 855–1296), we demonstrated that T cells of Balb/c (H-2^d) mice, primed with one injection of toxoid, recognized two major regions within residues 897–915 and 939–957. After multiple inoculations with toxoid, T cells of Balb/c expanded their recognition ability and responded very well to challenge with peptide 1261–1279 and moderately to stimulation with peptide 1149–1167. Unlike Balb/c T cells, those of toxoid-primed SJL (H-2^s) mice exhibited a more complex profile and responded to challenge with a large number of overlapping peptides. After one toxoid injection, however, three peptides, 897–915, 939–957/953–971 overlap and 1051–1069, were the most potent T cells stimulators. After three toxoid injections, peptides 897–915 and 1051–1069 remained immunodominant while the third region was shifted upstream to 925–943/939–957 overlap. The immunodominant epitope within peptide 897–915 was recognized exclusively by T cells, since no Abs were detected against this region. The Ab binding profiles of the two mouse strains were quite similar, showing only small quantitative differences. Both, Balb/c and SJL anti-toxoid Abs displayed strong binding mainly to peptide 1177–1195, followed by peptides 869–887/883–901 overlap and 1275–1296. In addition, a significant amount of Balb/c anti-toxoid Abs was bound to peptide 1135–1153. Unlike Balb/c Abs, that interacted weakly with peptides 995–1013 and 1051–1069, the anti-toxoid Abs of SJL mice exhibited strong binding toward both peptides. The results showed that, in a given strain, the regions recognized by anti-toxoid Abs and T cells may coincide or may be uniquely B or T cell determinants.