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.     

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.     

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2^b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2K^b and H-2D^b MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2K^b and H-2D^b and the predictive value of these motifs is shortly discussed. The high-affinity H-2D^b-binding peptide and putative CTL epitope E₇ 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E₇ 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.     

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.     

Major histocompatibility complex class I-restricted presentation of influenza virus nucleoprotein peptide by B lymphoma cells harboring an antibody gene antigenized with the virus peptide

We analyzed the capacity of B cells to process and present a peptide from the variable region of an endogenous immunoglobulin heavy (H) chain to a major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) clone. The H-chain gene was engineered to express 14-amino acid peptide from the sequence of the influenza virus nucleoprotein (NP) antigen in the third complementarity-determining region (CDR3). This NP peptide is presented in association with the D^b allele in H?2^b mice. We demonstrate that B lymphoma cells (H-2^b) harboring the antigenized H-chain gene process and present the NP peptide in association with the D^b molecule and are lysed by a CTL clone specific for that peptide in an MHC-restricted way. In contrast, the soluble antigenized antibody failed to mediate lysis of H?2^b target cells. The endogenously processed immunoglobulin CDR3 peptide could be eluted from surface D^b molecules in transfected cells. This study formally demonstrates that peptides from the hypervariable loops of endogenous immunoglobulin are processed through the endogenous degradative pathway and are presented to CD8⁺ T cells in the context of MHC class I molecules. The implication of these findings for processing and presentation of endogenous immunoglobulin peptides in B cells and network regulation by idiopeptides is discussed.     

Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2K^d to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2K^d. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2K^d-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2K^d; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2K^d. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2K^d to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2K^d class I molecule, and only a fraction of these serve as CTL epitopes.     

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.     

Glycosylphosphatidylinositol-linked Db does not induce an influenza-specific cytotoxic T lymphocyte response or recycle membrane-bound peptides

Major histocompatibility complex (MHC) class I molecules, as well as MHC class I-bound peptides, are known to recycle between the cell surface and an undefined, endosomal-like compartment. Little is known about the functional significance of this process. We have explored this using two different forms of the H-2D^b molecule expressed in transgenic mice, either transmembranous (D^b-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (D^b-GPI). The recycling capacity of peptides bound to D^b-tm and D^b-GPI was investigated using glycosylated D^b-binding glycopeptides, which were detected by flow cytometry. Only the tm form of D^b was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus (PR8) strain and tested for cytotoxic T lymphocyte (CTL) responses against an immunedominant nucleoprotein epitope (366–374, ASNENMETM), onyl D^b-tm mice were found to generate specific CTL responses. The results support the idea that membrane recycling of MHC class I-bound peptides on antigen-presenting cells may be important for the generation of certain CTL 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.     

Preferential usage of Vα4 and Vβ10 T cell receptor genes by lymphocytic choriomeningitis virus glycoprotein-specific H-2Db-restricted cytotoxic T cells

Correlations between the T cell receptor (TcR) V gene usage and the specificity of T cells have been primarily described for major histocompatibility complex (MHC) class II-restricted helper T cell responses. In the present study the TcR genes expressed by MHC class I-restricted murine cytotoxic T cells (CTL) specific for a major epitope of the lymphocytic choriomeningitis virus (LCMV), LCMV-GP2_275–289, were investigated. The TcR primary structure of an LCMV-GP2_275–289 specific H-2D^b-restricted CTL clone has been determined. It uses a member of the V_α4 family joined to J_αAN14.4 for the α chain and V_β10 rearranged to D_β2.1 and J_β2.4 for its β chain. Four other independent LCMV-GP2_275–289 specific H-2D^b-restricted CTL clones also expressed V_α4 and V_β10 gene elements. Furthermore, V_α4 and V_β10 were preferentially expressed by polyclonal CTL of C57BL/6 origin specific for LCMV. These results suggest that both TcR V_α and V_β regions are important for the recognition of the LCMV-GP2_275-289 epitope on H-2D^b molecules.     

Major histocompatibility complex class I allele-specific peptide libraries: Identification of peptides that mimic an H-Y T cell epitope

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2K^b-restricted octamers and H-2D^b-restricted nonamers. Peptides from the K^b-restricted library (K^bL: SXIXFXXL) and the D^b-restricted library (D^bL: XXXXNXXX^I_M) specifically stabilize empty K^b and D^b molecules, respectively. The libraries contain peptides that mimic several H-2^b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a D^b-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.     

A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells

This study identifies instability of MHC class I/peptide complexes and intermolecular competition for MHC class I presentation as factors responsible for the subdominance of cyto toxic T lymphocyte (CTL) epitopes. This evidence is based on the characterization of a new CTL epitope derived from the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV). This epitope, peptide GP117-125 (GP117) is presented to T cells by the mouse MHC class I molecule, H-2D^b. In short-term experiments induction of GP117-specific CTL by vaccination rendered C57BL/6 mice only partially resistant to infection with wild-type LCMV (LCMV-WE) but completely resistant to challenge with a previously described LCMV variant. The variant virus, LCMV-8.7B23, bears point mutations within both known LCMV-GP, H-2 D^b-restricted epitopes GP33-41 (GP33) and GP276-286 (GP276) resulting in a valine to leucine change at position 35 in peptide GP33 (V35L) and an asparagine to serine change at position 280 in peptide GP276 (N280S). Although variant peptide GP33/V35L stimulates a weak CTL response, GP276/N280S does not. Elution of peptide GP117 from both LCMV-WE- and LCMV-8.7B23-infected cells revealed that the difference in the capacity of GP117-specific CTL to protect against LCMV-WE and the virus variant LCMV-8.7B23 was due to differences in the level of GP117 presentation on the surface of both types of cells. Thus, it appears that the protective capacity of CTL specific for the subdominant epitope GP117 is influenced by the extent of presentation of other immunodominant peptide epitopes present within infected cells.     

Immunization with glycosylated Kb-binding peptides generates carbohydrate-specific,unrestricted cytotoxic T cells

Cytotoxic T cells (CTL) recognize target proteins as short peptides presented by major histocompatibility complex (MHC) class I restriction elements. However, there is also evidence for peptide-independent T cell receptor (TCR) recognition of target proteins and non-protein structures. How such T cell responses are generated is presently unclear. We generated carbohydrate (CHO)-specific, MHC-unrestricted CTL responses by coupling di- and trisaccharides to K^b- or D^b-binding peptides for direct immunization in mice. Four peptides and three CHO have been analyzed with the CHO either in terminal or central positions on the carrier peptide. With two of these glycopeptides, with galabiose (Galα1-4Gal; Gal₂) bound to a homocysteine (via an ethylene spacer arm) in position 4 or 6 in a vesicular stomatitis virus nucleoprotein-derived peptide (RGYVYQGL binding to K^b), CTL were generated which preferentially killed target cells treated with glycopeptide compared to those treated with the core peptide. Polyclonal CTL were also found to kill target cells expressing the same Gal₂ epitope in a glycolipid. By fractionation of CTL, preliminary data indicate that glycopeptide-specific K^b-restricted CTL and unrestricted CHO-specific CTL belong to different T cell populations with regard to TCR expression. The results demonstrate that hapten-specific unrestricted CTL responses can be generated with MHC class I-binding carrier peptides. Different models that might explain the generation of such responses are discussed.     

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen

Murine MHC class I-restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL-defined epitopes of this viral envelope protein are the L^d -binding 12-mer S28 – 39 peptide IPQSLDSWWTSL in H-2 ^d mice, and the K^b -binding 8-mer S208 – 215 peptide ILSPFLPL in H-2^b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H-2 class I-binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the L^d -binding peptide S28 – 39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the K^b -binding peptide S208 – 215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I-binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.     

Generation of hen egg lysozyme-specific and major histocompatibility complex class I-restricted cytolytic T lymphocytes: recognition of cytosolic and secreted antigen expressed by transfected cells

Syngeneic cells exogenously supplied with hen egg lysozyme (HEL) or endogenously synthesizing HEL were used as antigen-presenting cells to induce major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL). Immunization of C57BL/6 mice followed by repeated stimulation of their splenocytes in vitro with trypsinized HEL peptides led to the generation of CTL lines specific for trypsinized HEL peptides and restricted by H-2K^b. Immunization of C3H mice with a mixture of soluble native HEL and irradiated syngeneic spleen cells followed by in vitro stimulation of immune spleen cells with soluble HEL could in a few cases result in HEL-specific CTL able to kill syngeneic transfectant L cells secreting HEL (HELs) or expressing cytosol-targeted HEL (HELc). The use of HELs or HELc transfectant L cells as in vivo and in vitro immunogens was a potent way for eliciting HEL-specific polyclonal CTL. These CTL and two CD8⁺ clones were found to be H-2K^k restricted and specific for the 1-17 N-terminal HEL peptide. In addition, the anti-HEL CTL could also exhibit a significant cross-reactivity against unsensitized and HEL-untransfected targets expressing the K restriction element. This cross-reactivity was likely due to recognition of unidentified HEL mimicking peptides (self-derived ?) presented by the MHC class I (H-2K^b or H-2K^k) molecule used as the restriction element for the specific recognition of HEL. The CTL raised after immunization with HELs or HELc transfectant cells were found to recognize both the HELs and HELc transfectant cells even though HEL was not detected in the latter after a 2- or 5-min radiolabeling pulse. Recognition of both HELs and HELc transfectant cells by a given CTL clone suggests that HEL subjected to two separate processing pathways, each depending on the initial subcellular localization, can ensure the generation of similar MHC class I peptide complexes.     

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2D^b using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind D^b. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to D^b. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the D^b pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited D^b-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all D^b-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, D^b-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to D^b, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.     

Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries

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 I a molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1^c, which carries unusual key residues at both ends of the groove, and RT1.A^a 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-K^b, 8 – 13-mer and H2-D^b, 9 – 15-mer peptides). The rat MHC class Ia molecule, RT1-A^a, revealed a preference for 9 – 15-mer peptides, whereas RT1-A1^c 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.     

A wild-type p53 cytotoxic T cell epitope is presented by mouse hepatocarcinoma cells

The possibility to identify epitopes presented by tumor cells to cytotoxic T lymphocytes (CTL) has given rise to new fields in tumor immunology. The tumor suppressor gene product p53 is a good candidate antigen because it is involved in the tumorigenesis of many cancers. It accumulates in an inactivated form due to mutation or formation of heterodimers with an oncogene product. Epitopes from the mutant or wild-type p53 proteins are thought to be presented by tumor cells and to induce a tumor-specific CTL response. To identify such epitopes, mouse wild-type p53 peptides encompassing the H-2 D^b anchoring motif were tested for their association with the D^b molecule. Positive peptides were assayed for their ability to induce CTL in C57BL/6 mice. CTL specific for one wild-type p53 peptide, p232–240, were isolated and found to lyse hepatocarcinoma cell lines established from mice transgenic for simian virus 40 large T antigen which overexpress p53. These results show that the p232–240 epitope from wild-type p53 is naturally processed and presented in H-2^b tumor cells.     

Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: Implications on immunogenicity and immunodominance

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA-A*0201/K^b (A2/K^b) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA-A*0201-binding motif, we selected A2-binding peptides, immunized A2/K^b animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide-specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/K^b animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide-specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.     

Cytotoxic T lymphocytes specific for murine type II collagen do not trigger arthritis in B10 mice

To investigate the role of cytotoxic T lymphocytes (CTL) in arthritis, we set out to induce CTL specific for murine type II collagen (mCII) in a mouse model. The primary protein sequence of the murine pro-α1(II) was screened for fragments bearing H-2 D^b or K^b binding motifs. Six fragments were identified and the corresponding peptides synthesized. One of these peptides, peptide P201 (amino acid 199–208 in the C-propeptide of the murine pro-α1(II)), was found to be a strong binder to H-2 D^b. When used to treat RMA-S cells at 26°C, peptide P201 induced a four-fold increase of surface expression of H-2 D^b. Administration of the P201-treated RMA-S cells into B10 mice (H-2^b) induced strong CTL responses against the immunizing collagen peptide. Despite the high frequencies of mCII-specific CTL precursors in the periphery, however, the immunized mice showed no sign of arthritis up to 16 weeks after immunization. Implications of these data for autoimmunity and arthritis are discussed.