Chemical and functional analysis of MHC class II-restricted T cell epitopes.

Various aspects of antigen degradation and presentation are reviewed, in particular with respect to fragmentation of native vs. denatured proteins, different enzymatic machinery present in different cells and individuals, characterization of epitopes and their persistence on antigen-presenting cells as well as their capacity to interact with different MHC class II molecules. Finally, the structure of antigenic peptides is discussed.     

Structural basis for the recognition of mutant self by a tumor-specific, MHC class II-restricted T cell receptor

Structural studies of complexes of T cell receptor (TCR) and peptide-major histocompatibility complex (MHC) have focused on TCRs specific for foreign antigens or native self. An unexplored category of TCRs includes those specific for self determinants bearing alterations resulting from disease, notably cancer. We determined here the structure of a human melanoma-specific TCR (E8) bound to the MHC molecule HLA-DR1 and an epitope from mutant triosephosphate isomerase. The structure had features intermediate between 'anti-foreign' and autoimmune TCR-peptide-MHC class II complexes that may reflect the hybrid nature of altered self. E8 manifested very low affinity for mutant triosephosphate isomerase-HLA-DR1 despite the highly tumor-reactive properties of E8 cells. A second TCR (G4) had even lower affinity but underwent peptide-specific formation of dimers, suggesting this as a mechanism for enhancing low-affinity TCR-peptide-MHC interactions for T cell activation.     

T cell receptor recognition of a 'super-bulged' major histocompatibility complex class I-bound peptide

Unusually long major histocompatibility complex (MHC) class I-restricted epitopes are important in immunity, but their 'bulged' conformation represents a potential obstacle to alphabeta T cell receptor (TCR)-MHC class I docking. To elucidate how such recognition is achieved while still preserving MHC restriction, we have determined here the structure of a TCR in complex with HLA-B(*)3508 presenting a peptide 13 amino acids in length. This complex was atypical of TCR-peptide-MHC class I interactions, being dominated at the interface by peptide-mediated interactions. The TCR assumed two distinct orientations, swiveling on top of the centrally bulged, rigid peptide such that only limited contacts were made with MHC class I. Although the TCR-peptide recognition resembled an antibody-antigen interaction, the TCR-MHC class I contacts defined a minimal 'generic footprint' of MHC-restriction. Thus our findings simultaneously demonstrate the considerable adaptability of the TCR and the 'shape' of MHC restriction.     

T cell recognition of Tn-glycosylated peptide antigens

The mouse hemoglobin-derived decapeptide Hb (67–76), VITAFNEGLK, which binds well to E^k and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (α-D -N-acetylgalactosamine, or α-D -GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to E^k and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to E^k. This was the case for the glycopeptides with Tn attacted at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to E^k, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67–76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.     

The Yersinia enterocolitica invasin protein promotes major histocompatibility complex class I- and class II-restricted T-cell responses

Yersinia enterocolitica invasin (Inv) protein confers internalization into and expression of proinflammatory cytokines by host cells. Both events require binding of Inv to beta1 integrins, which initiates signaling cascades including activation of focal adhesion complexes, Rac1, mitogen-activated protein kinase, and NF-kappaB. Here we tested whether Inv might be suitable as a delivery molecule and adjuvant if used as a component of a vaccine. For this purpose, hybrid proteins composed of Inv and ovalbumin (OVA) were prepared, applied as a coating to microparticles, and used for vaccination. Fusion of OVA to Inv did not significantly disturb the ability of Inv to promote host cell binding, internalization, and interleukin-8 (IL-8) secretion when applied as a coating to microparticles. The microparticles were used for vaccination of mice adoptively transferred with OVA-specific T cells from OT-1 or DO11.10 mice. Administration of OVA-Inv-coated microparticles induced OVA-specific T-cell responses. OVA-specific CD4 T cells produced both gamma interferon (IFN-gamma) and IL-4 as determined by enzyme-linked immunosorbent assay. Likewise, pronounced OVA-specific CD8 T-cell responses associated with IFN-gamma production were observed. Together, these results suggest that Inv might be an attractive tool in vaccination as it confers both host cell uptake and adjuvant activity by engagement of beta1 integrins of host cells, which leads to CD4 as well as CD8 T-cell responses.     

Precursors of functional MHC class I- or class II-restricted CD8alphaalpha(+) T cells are positively selected in the thymus by agonist self-peptides

The origin and specificity of alphabeta TCR(+) T cells that express CD8alphaalpha have been controversial issues. Here we provide direct evidence that precursors of functional CD8alphaalpha T cells are positively selected in the thymus in the presence of agonist self-peptides. Like conventional positive selection, this agonist selection process requires functional TCR alpha-CPM, whereas it is independent of CD8beta expression. Furthermore, CD8alphaalpha expression on mature, agonist-selected T cells does not imply selection by MHC class I, and CD8alphaalpha(+) T cells can be either class I or class II restricted. Our data define a distinct agonist-dependent, positive selection process in the thymus, and they suggest a function for CD8alphaalpha distinct from the conventional TCR coreceptor function of CD8alphabeta or CD4.     

The identification of HLA class II-restricted T cell epitopes to vaccinia virus membrane proteins

Three decades after the eradication of smallpox, the threat of bioterrorism and outbreaks of emerging diseases such as monkeypox have renewed interest in the development of safe and effective next-generation poxvirus vaccines and biodefense research. Current smallpox vaccines contain live virus and are contraindicated for a large percentage of the population. Safer, yet still effective inactivated and subunit vaccines are needed, and epitope identification is an essential step in the development of these subunit vaccines. In this study we focused on 4 vaccinia membrane proteins known to be targeted by humoral responses in vaccinees. In spite of the narrow focus of the study we identified 36 T cell epitopes, and provide additional support for the physical linkage between T and B epitopes. This information may prove useful in peptide and protein-based subunit vaccine development as well as in the study of CD4 responses to poxviruses.     

Similar structural requirements for T cell and antibody recognition of an MHC class I:peptide complex

We have generated a monoclonal antibody (Fab 13.4.1) using the phage display technique, which recognizes the murine MHC class I molecule, K^k, in complex with the influenza virus derived peptide, HA255-262, but not in complex with other peptides. Using BIAcore analysis the kinetics of the antibody was used to calculate the K_D to be about 10⁻⁹ M, which is typical of an antibody, and several orders of magnitude higher than the corresponding value for T cell receptors.

Given the specificity and affinity of Fab13.4.1 we expected it to inhibit K^k restricted and HA255-262 specific T cell hybridomas, but not K^k restricted T cells with different specificity, which indeed was found to be the case. Thus, Fab13.4.1, recognizes a K^k:HA255-262 dependent epitope which is related to the epitope recognized by the specific T cells. A more detailed analysis of the structural requirements for T cell and antibody recognition of the K^k:HA255-262 complex revealed that they use similar contacts points to interact with this MHC class I:peptide complex.     

Processing of viral antigens and presentation to class II-restricted T cells

Some antigens require intracellular processing by antigen presenting cells before being presented to T cells in conjunction with surface major histocompatibility complex antigens. The whole mechanism of these processing events is not known and in this article, Kingston Mills puts forward arguments for alternative routes of antigen processing, with particular reference to recognition of viral proteins by class II-restricted T-cell clones.     

Synthetic peptide libraries in the determination of T cell epitopes and peptide binding specificity of class I molecules.

Major histocompatibility complex (MHC) class I molecules combine with short peptides of defined length and sequence. Here we describe an approach that may be used in the analysis of peptide preference of different allelic MHC class I molecules, and in the determination of T cell epitopes. We produced synthetic "peptide libraries" of limited complexity by standard peptide chemistry. Using these peptide mixtures we show that H-2 Kb molecules can accommodate both 8- and 9-residue peptides, whereas Db molecules are unable to combine with peptides shorter than 9 amino acids present in these libraries. When these peptide mixtures are used to provide "fingerprints" of Db molecules and mutants thereof, both loss and gain of the ability to combine with certain peptides is observed. For the Kbm1 mutant a strong influence of amino acid substitutions in class I molecules on the peptides selected is observed. In these synthetic peptide mixtures, the presence of a specific T cell epitope, known to be represented once, can be detected. This approach may be extended to the identification of new T cell epitopes from larger peptide libraries.     

N-linked carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4(+) T cells

Glycosylation of mammalian proteins is known to influence their intracellular trafficking, half life, and susceptibility to enzymatic degradation. Rare instances of natural T cell epitopes dependent upon glycosylation for recognition have been described. We report here on human CD4(+) T lymphocyte cultures and clones from two melanoma patients that recognize the melanoma-associated Ag tyrosinase in the context of HLA-DR4 and -DR8. These T cells recognize tyrosinase, normally a heavily glycosylated molecule, when expressed constitutively in melanoma cells or in COS-7 transfectants pulsed as lysates onto autologous APC. However, these T cells fail to recognize tyrosinase expressed in bacteria, nor do they react with overlapping peptides covering full-length tyrosinase, suggesting a critical role for glycosylation in the processing and / or composition of the stimulatory epitopes. The requirement for glycosylation was demonstrated by the failure of tyrosinase-specific CD4(+) T cells to recognize tyrosinase synthesized in the presence of glycosylation inhibitors, or deglycosylated enzymatically. Site-directed mutagenesis of each of seven potential N-glycosylation sites showed that four sites were required to generate forms of tyrosinase that could be recognized by individual T cell clones. These data indicate that certain carbohydrate moieties are required for processing the tyrosinase peptides recognized by CD4(+) T cells. Post-translational modifications of human tumor-associated proteins such as tyrosinase could be a critical factor for the development of antitumor immune responses.     

Functional degeneracy of residues in a T cell peptide epitope contributes to its recognition by different T cell hybridomas.

Synthetic antigen Poly EYK(EYA)5 induces T cells of narrowly defined fine specificity as represented by the two I-Ad-restricted T cell hybridomas, A.1.1 and B.1.1. Both these hybridomas recognize the minimum 15-amino-acid peptide sequence EYK(EYA)4. We have characterized the residues involved in the recognition of EYK(EYA)4 peptide by these hybridomas with synthetic peptides and discovered a distinct functional hierarchy for the residues in the sequence. Even with the repeating tripeptide (EYA)5, which is recognized by B.1.1 cells, the residues that are essential cluster near the middle of the sequence but not near the N- or C-terminal region. Different MHC binding and TCR contacting residues were found for each of the hybridomas. The results suggest that different T cells either recognize different parts of the peptide MHC complex or that the peptide binds to MHC in multiple conformations. This was supported by the fact that Poly EYK(EYA)5 is alpha-helical but the peptides used here showed only a slight propensity to adopt this structure and it did not correlate with their functional activity. We also found that (EYA)5 does not compete with EYK(EYA)4 in the stimulation of A.1.1 cells despite its obvious capacity to interact with I-Ad when it stimulates B.1.1 cells. This may be because these peptides have a low affinity for Ia and therefore only appropriate TCR interactions would stabilize the antigen-Ia complex. In conclusion, antigen-MHC-TCR interaction appears to be a dynamic process which allows recognition of different residues of a T cell determinant by different T cells.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a “promiscuous” peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.     

Specific lysis of Listeria monocytogenes-infected macrophages by class II-restricted L3T4+ T cells

Mice were infected with the intracellular bacterium, Listeria monocytogenes, and T cell clones from spleens, lymph nodes and peritoneal exudates were established. The capacity of L3T4⁺, Lyt2^? T-cell clones to specifically lyse L. monocytogenes-infected macrophages was analyzed. As a source of target cells, bone marrow macrophages (BMMΦ) after 9 days of culture in hydrophobic teflon bags were used. These BMMΦ were totally Ia^?; however, significant Ia-expression could be induced by interferon-γ (IFN-γ). IFN-γ-stimulated BMMΦ, after priming with live or killed L. monocytogenes organisms were effectively lysed by the vast majority of L3T4⁺ T cell clones. In the absence of either IFN-γ stimulation or antigen priming, no lysis occurred. Cytolysis was demonstrable in a conventional 4-h ⁵¹Cr-release assay and in an 18-h neutral red uptake assay and was antigen specific and class II restricted. Native T cells from L. monocytogenes-infected mice failed to lyse stimulated, L. monocytogenes-primed BMMΦ and gained their cytolytic activity after antigenic restimulation in vitro. These data demonstrate that L. monocytogenes-specific L3T4⁺ T cells could lyse MΦ presenting listerial antigens provided that Ia antigen expression had been induced. L3T4⁺ T cell clones produced IFN-γ after restimulation with antigen plus accessory cells in vitro and IFN-γ secretion could be increased by costimulation with recombinant IL 2. These T cell clones conferred significant protection upon recipient mice which was more pronounced in the liver. The possible relevance of lysis by L3T4⁺ T cells of infected MΦ to protection against and pathogenesis of intracellular bacterial infections is discussed.     

T cell recognition of donor major histocompatibility complex class I peptides during allograft rejection.

LEW (RTI1) recipients of DA (RTIav1) skin and kidney allografts were tested for the capacity of their T lymphocytes to proliferate to three 22-24-amino acid peptides from the hypervariable regions of the RTI-Aav1 classical MHC class I molecule. Ten days after rejecting second-set DA kidney allografts, spleen cells (but interestingly not lymph node cells) from LEW recipients showed strong, LEW antigen-presenting cell-dependent, CD4+ T cell proliferation to peptide 1 (from the alpha helical region of the alpha 1 domain). CD8+ T cells showed no response to peptide 1. There was no response by the spleen cells to peptide 2 (from the beta sheet of the alpha 2 domain) or peptide 3 (from the alpha helical region of the alpha 2 domain). Immunization of LEW rats with pure RTI-Aav1 class I H chain in Freund's adjuvant gave responses identical to that seen after grafting, i.e. good CD4+ T cell proliferation to peptide 1, but none to peptides 2 and 3. However, immunization of LEW rats with peptides 1, 2 and 3 in Freund's adjuvant resulted in good CD4+ T cell proliferation responses to each of the peptides. These data demonstrate that indirect allorecognition can be stimulated by allograft rejection, and emphasize that the physiological processing of donor antigens will influence which peptides will be important in indirect allorecognition in transplantation.     

Enhancing antitumor immune responses: intracellular peptide delivery and identification of MHC class II-restricted tumor antigens

Summary: The importance of T‐cell‐mediated antitumor immunity has been demonstrated in both animal models and human cancer therapy. The identification of major histocompatibility complex (MHC) class I‐restricted tumor antigens has generated a resurgence of interest in immunotherapy for cancer. However, recent studies suggest that therapeutic strategies that have mainly focused on the use of CD8⁺ T cells (and MHC class I‐restricted tumor antigens) may not be effective in eliminating cancer cells in patients. Novel strategies have been developed for enhancing T‐cell responses against cancer by prolonging antigen presentation of dendritic cells to T cells and the inclusion of MHC class II‐restricted tumor antigens. identification of MHC class II‐restricted tumor antigens, which are capable of stimulating CD4⁺ T cells, not only aids our understanding of the host immune responses against cancer antigens, but also provides opportunities for developing effective cancer vaccines.     

Degenerate recognition of T cell epitopes: impact of T cell receptor reserve and stability of peptide:MHC complexes

The concept of molecular mimicry suggests that microbial pathogens might activate antigen-specific T cells that then cross-react with endogenous antigens and result in the generation of autoimmunity. Here we discuss several under-represented factors impacting the ability of TCRs to recognize a wide spectrum of related peptide:MHC (pMHC) ligands. Two of these factors include the affinity of the peptide for MHC and the level of TCR expression. Thymocytes that recognize peptides with low affinity for MHC avoid negative selection, but mature T cells, by virtue of increased TCR expression, will proliferate in response to these same unstable pMHC complexes. While the expression of a reserve of antigen receptors expands the potential number of epitopes for which a T cell can cross-react, phosphatase activity provides a tuning mechanism to increase the threshold level of activation. Thus, degenerate recognition of T cell epitopes involves the stability of the peptide:MHC complex, the number of TCR expressed, and the level of phosphatase activity.     

MHC class II peptide flanking residues of exogenous antigens influence recognition by autoreactive T cells

Molecular mimicry between exogenous microbial antigens and self-epitopes has been proposed as a triggering mechanism for autoimmune diseases for many years. We reported that a peptide from a protein specific to Chlamydia pneumoniae (Cpn0483) which shares a motif with the dominant encephalitogenic epitope of the self-antigen, rat myelin basic protein (rat68-86), elicits experimental autoimmune encephalomyelitis (EAE) in Lewis rats. We recently observed that rat68-86 utilizes aspartic acid (D) and arginine (R) in the common motif as primary and secondary TCR contacts, respectively. In contrast, the encephalitogenic activity of Cpn0483 is dependent on R and the C-terminal asparagine (N), which flanks the MHC class II-P9 anchor residue. Thus, rat68-86 and Cpn0483 share a common motif, are encephalitogenic and are both restricted by MHC class II RT1.B(l). T cells from rats immunized with the encephalitogenic Cpn0483 peptide proliferated to the priming peptide as well as to the non-encephalitogenic CpnN>A analog. However, CpnN>A-primed T cells did not respond to the native Cpn0483 peptide. We conclude that the MHC-flanking C-terminal asparagine residue markedly influences T cell recognition by the chlamydial peptide.