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.     

Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells

Allospecific immune responses against the MHC of another individual are remarkably strong, due t a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H-2b-derived murine lymphoma mutant RMA-S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA-S. Up-regulation of the levels of H-2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.     

Restriction of self-antigen presentation to cytolytic T lymphocytes by mouse peptide pumps

Transport of an immunogenic self-peptide from the second domain of the mouse major histocompatibility complex (MHC) H-2K^d class I molecule is blocked at the TAP1-TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1-TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self-peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies.     

Cytotoxic T lymphocyte recognition of HLA-A2 antigens in normal and HLA-Cw3-transgenic mice

It is well established that a large fraction of murine cytotoxic T cells can recognize allogeneic major histocompatibility complex (MHC) antigens, and that the majority of this response are not restricted by H-2 antigens of the responding host. In contrast, the murine response against the xenogeneic HLA class I antigens is relatively weak. Moreover, a large proportion of the responding murine T cells do not recognize the HLA antigen per se but only in an H-2-restricted manner, probably as an HLA peptide bound to H-2. Considerable evidence suggests that in mice the T cell repertoire is selected by thymic H-2 antigens. Therefore, we asked the question whether in transgenic mice expressing an HLA class I antigen the T cell repertoire would be shaped toward a more effective recognition of other HLA alleles. Normal C57BL/6 (B6) and HLA-Cw3-transgenic B6 mice were immunized with a B6-derived cell line transfected with HLA-A2. The resulting A2-specific CTL were tested on L cells transfected with either A2 alone, which should identify the H-2-unrestricted CTL, and on L cells transfected with A2 plus H-2b genes, which should identify the sum of H-2b-restricted and unrestricted CTL. Both bulk culture and limiting dilution experiments showed that the CTL precursor frequencies for A2-specific CTL were not increased in the transgenic mice, and that both strains produced comparable proportions of H-2b-restricted and of unrestricted A2-specific CTL. The B6.Cw3 mice seemed to respond less well to HLA-A2 than the normal B6 mice, suggesting the possibility of tolerance for peptides shared by the Cw3 and A2 molecules. In conclusion, the T cells in the B6.Cw3 transgenic mice did not seem to be selected towards a stronger and more unrestricted recognition of an allo-HLA antigen. The possible reasons are discussed.     

Influenza basic polymerase 2 peptides are recognized by influenza nucleoprotein-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) play an important role in limiting viral infections and in eradicating virus from host tissues. Recent progress in understanding the processing and presentation of viral antigens to CTL indicates that the CTL antigen receptor recognizes peptides derived from viral proteins that are bound to an antigen binding groove present in class I major histocompatibility complex (MHC) molecules. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccinia recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that while screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.     

Major histocompatibility complex binding and T cell recognition of a viral nonapeptide containing a minimal tetrapeptide.

The primary immune response of cytotoxic T lymphocytes in H-2d and H-2q mice to infection with lymphocytic choriomeningitis virus is directed mostly towards the common major T cell epitope of amino acids 112-132 on the viral nucleoprotein (NP). The molecules responsible for presentation of the T cell epitope NP112-132 are in both haplotypes the MHC class I L antigens (Ld, Lq). Truncations of the amino and carboxy termini of the NP 112-132 sequence revealed the nonapeptide RPQASGVYM (NP118-126) as a most effective peptide antigen, but even the tetrapeptide GVYM was recognized by CTL of both haplotypes in a class I antigen-restricted specificity. When tyrosine (Y) or methionine (M) were substituted with alanine, CTL recognition of the altered nonamer required 10(6) to 10(8) times higher peptide concentrations and in one case (Y----A on Ld) the peptide was not recognized at all. Up-modulation of the expression of Ld and Lq class I antigens as measured by flow cytometry correlated with the ability to present the peptide antigens. The only exception was peptide NP118-126 (M----A), which was recognized by T cells on L-Ld and L-Lq target cells but failed to up-regulate Ld and Lq antigens.     

Cytotoxic T lymphocytes against the antigen-processing-defective RMA-S tumor cell line.

RMA-S is an antigen processing-defective cell line, obtained from a Rauscher virus-induced tumor. The cells express only a low level of cell surface major histocompatibility complex (MHC) class I molecules, which are supposed to be devoid of internally derived antigenic peptides. We investigated Rauscher virus expression and Rauscher peptide presentation to virus-specific cytotoxic T lymphocytes (CTL) by this cell line. Viral proteins are expressed properly, both intracellularly and at the cell surface of RMA-S. Rauscher peptides are presented to virus-specific CTL in the groove of both the class I H-2Kb and Db molecules, but at a low level. Culture of RMA-S cells at room temperature increases their susceptibility to CTL. The RMA-S defect thus affects, but not totally abrogates, Rauscher peptide presentation by MHC class I molecules via the endogenous pathway. This indicates that the RMA-S antigen processing deficit is not absolute.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

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.     

The assembly of functional beta(2)-microglobulin-free MHC class I molecules that interact with peptides and CD8(+) T lymphocytes

Functional MHC class I molecules are expressed on the cell surface in the absence of beta(2)-microglobulin (beta(2)m) light chain that can interact with CD8(+) T lymphocytes. Whether their assembly requires peptide binding and whether their recognition by CD8(+) T lymphocytes involves the presentation of peptide epitopes remains unknown. We show that beta(2)m-free H-2D(b) assembles with short peptides that are approximately 9 amino acid residues in length, akin to ligands associated with completely assembled beta(2)m(+) H-2D(b). Remarkably, a subset of the peptides associated with the beta(2)m-free H-2D(b) has an altered anchor motif. However, they also include peptides that contain a beta(2)m(+)H-2D(b) binding anchor motif. Further, the H-2K(b)- and H-2D(b)-restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2(b) class I in beta(2)m-deficient cells and are recognized by epitope-specific CD8(+) T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of beta(2)m with peptides and form CD8(+) T lymphocyte epitopes.     

Differential processing of influenza nucleoprotein in human and mouse cells

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265 – 273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265 – 273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.     

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 K^d. 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 K^d 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/K^d complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.     

Peptide loading of empty major histocompatibility complex molecules on RMA-S cells allows the induction of primary cytotoxic T lymphocyte responses.

The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

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.     

Interaction of In Vitro- and In Vivo-Generated Cytotoxic T Cells with SV40 T Antigen: Analysis with Synthetic Peptides

Virus-specific cytotoxic T cells recognize antigens in the form of peptides (8 or 9 amino acids long) bound to MHC class-I molecules. Exposure of unprimed murine splenocytes to synthetic peptides of viral antigens elicits primary CTL in vitro. The fme specificity of such CTL as well as the correlation between binding affinity of peptides to class-I molecules and CTL induction was analysed using synthetic peptides corresponding to overlapping and distinct amino-acid residues in SV40 T antigen (Tag) D_b-restricted T-cell epitopes I, II-III, and V. The peptides induced cross-reactive CD8₊ primary CTL in spienocytes of naive C57 BL/6 mice. This reactivity was seen regardless of the peptides allelic anchor motifs or their abilities to stabilize empty class-I molecules. However, none of the primary CTL and CTL lines lysed Tag-expressing cells. In contrast, CTL generated in vivo by immunizing mice with Tag-expressing cells recognized endogenously processed Tag as well as synthetic peptides. The peptides recognized by these CTL depended on the intracellular concentration of Tag antigen in the immunizing cells. The reactivity of these CTL was peptide specific as shown by a functional peptide competition assay. Moreover, three peptides bound to and were recognized in the context of both K^b and D^b molecules. These results have revealed a flexible disposition of MHC class-I molecules with regard to peptide binding and also reflected lack of correlation between binding affinity to class-1 molecules and the capacity of peptides to induce primary CTL or to serve as potential targets. The significance of these findings in relation to identifying major T-cell epitopes using allele specific peptide motif and in vitro maintained CTL clones is discussed.     

Restoration of MHC Class I Surface Expression and Endogenous Antigen Presentation by a Molecular Chaperone

Presentation of cytosolic peptides in the context of major histocompatibility complex (MHC) class I antigen is crucial for immune recognition of virus-infected and malignant cells. This process, which is often defective in cancer cells, involves a series of cellular events which may be facilitated by heat shock proteins (molecular chaperones). To address the influence of chaperone function on the presentation of cytosolic peptides, we have utilized B16 melanoma cells (H-2^b). These tumour cells are resistant to lysis by MHC class I-restricted cytotoxic T lymphocytes (CTL), due to a very low level of surface MHC expression. The authors found that stably transfected clones of B16 expressing a heterologous heat shock protein (Hsp65) exhibit significantly increased levels of MHC class I antigens on their surface, and are effectively lysed by alloreactive CTL. These MHC class I molecules can form functional MHC-peptide complexes which are recognized by virus-specific CTL. Moreover, mice immunized with Hsp65-expressing tumour cells, but not with control-transfected tumour cells, display a significantly increased resistance to a subsequent challenge with live, wild-type B16. Together, these results indicate that the suitable expression of a molecular chaperone can overcome a defect in MHC class I expression and antigen presentation, and suggest a novel approach to cancer immunotherapy.     

Cytotoxic T lymphocyte responses to wild-type and mutant mouse p53 peptides

Cytotoxic T lymphocytes (CTL) recognize peptides presented at the cell surface in association with major histocompatibility complex (MHC) class I molecules. The finding that peptides binding to MHC class I molecules share common amino acid motifs renders feasible the selection of antigenic peptides by simply scanning protein sequences, and thus, provides the possibility of inducing CTL to pre-defined specificities. Tumor cells possess antigens known to generate MHC class I-restricted CD8⁺ CTL responses. Thus, these antigens represent good targets to induce tumor-specific immunity. Among these antigens, the p53 tumor suppressor gene product is an attractive candidate for cancer immunotherapy. Mutations in the p53 gene have been found to be very frequently associated with a malignant transformation and often lead to p53 protein overexpression. Thus, we investigated the possibility of inducing CTL to wild-type or mutant p53 peptides in a BALB/c (H-2^d) mouse model. Peptides possessing the H2-K^d binding motif were selected and tested for binding to the H-2K^d molecules in vitro. Synthetic peptides p53_122–130 wild-type or “mutant” (Lys → Glu substitution at position 129) were shown to be the best binder peptides and were tested for their immunogenicity in mice. H-2K^d-restricted p53-specific CD8⁺ CTL were generated following immunization of mice with either wild-type (wt) p53_122–130 or mutant (mut) p53_122–130 (E129) peptides. Only low-affinity CTL can be obtained by immunization with the wt sequence. In contrast, CTL elicited with the mut peptide recognized the mut sequence at a 10–100-fold lower concentration. This indicates that CTL elicited with the mut peptide recognized the mut sequence very efficiently, whereas the wt sequence is poorly recognized, if at all. Taken together, these results thus suggest that p53-specific tumor immunotherapy may be successful only if the mutated protein is taken into consideration.     

Antigen processing mutant T2 cells present viral antigen restricted through H-2K

Cytotoxic T lymphocytes (CTL) recognize foreign antigens as short peptides presented by class I molecules of the major histocompatibility complex (MHC). T2 cells are profoundly defective in the presentation of endogenously synthesized antigens to CTL due to a deletion of MHC class II-encoded genes for transporters associated with antigen presentation (TAP1/TAP2). Surprisingly, we here demonstrate that T2 cells, after infection with Sendai virus, are readily killed by H-2K^b restricted CD8⁺ T cells. In contrast to classical class I-mediated antigen presentation, the presentation of Sendai virus antigen inT2K^b cells is brefeldin A (BFA) insensitive. The present findings may suggest the presence of an alternative pathway for MHC class I-mediated antigen presentation in T2 cells.     

Thymus leukemia antigen (TL)-specific cytotoxic T lymphocytes recognize the alpha1/alpha2 domain of TL free from antigenic peptides

The thymus leukemia antigens (TL) belong to the MHC class Ib family and can be recognized by CD8-dependent or -independent cytotoxic T lymphocytes (CTL) showing TL, but not H-2, restriction. We previously reported that the CTL epitope is TAP independent and in the present study we further characterize the recognition mechanism of CD8-dependent TL-specific TCRalphabeta CTL. We first prepared empty TL tetramers by way of peptide-independent folding with recombinant proteins produced in an Escherichia coli expression system, and showed that TL-specific CTL recognized TL without putative TL-associated peptide and/or post-translational modifications of TL by mammalian and insect cells. We next prepared transfectants expressing various chimeric TL molecules with mouse or human MHC class I as well as chimeric TL tetramers with recombinant proteins produced by insect cells, and demonstrated that chimeric TL whose alpha3 domain was replaced by that of H-2K(b), but not of HLA-A2, was sufficient for binding and activation of TL-specific CTL. These results indicate that TL-specific CTL recognize predominantly their alpha1/alpha2 domain as an epitope(s) and that the binding activity to the murine CD8 of the alpha3 domain of H-2K(b) is sufficient to induce their CTL activity, although it is known to be weaker than that of TL, but stronger than that of HLA. The results taken together indicate that CD8-dependent TL-specific TCRalphabeta CTL recognize an epitope(s) of the alpha1/alpha2 domain of TL free from antigenic molecules, and that CD8 plays an important role in stable interactions between TL and their corresponding TCR.