Mapping of epitopes recognized by alloreactive cytotoxic T lymphocytes using inhibition by MHC peptides

To identify epitopes recognized by alloreactive CTL we have examined H-2Kb-specific CTL for their recognition of synthetic peptides with sequences derived from the native Kb class I molecule. Consecutive nested peptides spanning the immunogenic alpha 1 and alpha 2 domains of Kb were tested for their capacity to inhibit CTL clones in their recognition of cells expressing the native Kb molecule. Inhibition by these peptides was found to be an extremely rare event. One peptide (Kb.111-122) did inhibit recognition by one particular CTL clone, clone 13. Upon further investigation it was observed that clone 13 also recognized peptide Kb.111-122 when presented in the context of the syngeneic MHC molecule, Kd. Considering that residues 111 to 122 are located at the base of the antigen groove, and clone 13 is able to recognize Kb.111-122 when presented by syngeneic target cells, we suggest that inhibition of this CTL clone may be due to MHC restricted, self-presentation of peptide rather than to direct binding of free peptide to the TCR. Taken together, these results suggest inhibition of allospecific CTL by MHC peptides is a rare event at least for Kb recognition. Furthermore, they demonstrate the need for caution when interpreting inhibition by peptide as evidence for recognition by the TCR of the corresponding region on the native molecule.     

Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein.

Epitope mapping of a MHC class I-restricted cytotoxic T cell response to nef, a regulatory protein of HIV, was performed with fresh PBMC from HIV-seropositive donors and target cells pulsed with a panel of overlapping peptides of the nef protein. These nef-specific CTL recognized a synthetic peptide of 10 residues derived from a nonamphipathic, highly conserved region of the nef protein in association with the HLA A3.1 molecule. Using human cell transfectants expressing mutations of the A3 molecule, we demonstrated that the amino acid at position 152 of the A3.1 molecule appears to be critical for detection of this response. Thus, rapid analysis of the epitopes of HIV proteins stimulating CTL responses can be achieved using a combination of fresh donor PBMC and target cells pulsed with synthesized peptides.     

Poliovirus-specific major histocompatibility complex class I-restricted cytolytic T-cell epitopes in mice localize to neutralizing antigenic regions.

A major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) response is induced in BALB/c mice upon immunization with poliovirus serotype 1 (Mahoney strain). A similar class I-restricted response is also induced upon immunization with purified VP1 capsid proteins. Thus, poliovirus-specific MHC class I CTL responses can be induced independently of viral infection in murine hosts. In experiments using recombinant vaccinia virus vectors expressing different segments of the poliovirus capsid proteins and synthetic peptides, two regions of the VP1 capsid protein appear to contain epitopes recognized by this bulk CTL population. These epitope regions contain a Kd-restricted peptide-binding motif. Interestingly, each of these CTL epitopes is located near previously defined neutralizing antigenic sites.     

Defining MHC class II T helper epitopes for WT1 tumor antigen

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4⁺ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1_124–138 and WT1_247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.     

Induction of cytotoxic T lymphocytes in mice against the principal neutralizing domain of HIV-1 by immunization with an engineered T-cytotoxic-T-helper synthetic peptide construct.

Peptide constructs were engineered by colinear synthesis of two short synthetic peptide determinants; a determinant recognized by T helper cells (TDh) and a determinant recognized by T cytotoxic cells (TDc). Three types of constructs were synthesized: TDc-TDh, TDh-TDc, and TDh-KK-TDc, where KK are two lysine residues. In vivo immunization with free construct induced cytolytic lymphocytes (CTL) only in the case of TDc-TDh. However, immunization with spleen cells to which these constructs had been internalized by hypertonic shock, induced CTL activity in all three cases. No CTL could be induced after immunization with free TDc in either protocol. These results indicate that cell internalization of the construct might be essential for CTL induction, and also, that "help" from the TDh seems to be required.     

Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes.

CTL constitute an essential part of the immune response against the HIV. CTL recognize peptides derived from viral proteins together with the MHC class I molecules on the surface of infected cells. The CTL response could be important in prevention or control of infection with HIV by destroying virus-producing cells. In this study we have attempted to identify peptide epitopes recognized by HIV-specific CTL. Using synthetic peptides, we have identified six conserved peptidic epitopes on the gp120 envelope glycoprotein recognized by polyclonal human CTL in association with HLA-A2 class I transplantation Ag. These results were confirmed by two approaches: i) blocking of CTL activities with antibodies specific for three of these conserved peptides; and ii) construction of doubly transfected P815-A2 target cells, using deletions of the HIV env gene. Vaccination or immunotherapy in HLA-A2 individuals can thus be considered using highly conserved HIV env peptidic sequences.     

Competition between unrelated peptides recognized by H-2-Kd restricted T cells

P815 (H-2d) target cells incubated with synthetic peptides corresponding to region 170-182 of HLA or to region 141-161 of influenza nucleoprotein (NP) are lysed by DBA/2 derived cytolytic T cells (CTL) specific for HLA or by BALB/c derived CTL-specific for NP, respectively. Both peptide Ag are recognized in the context of Kd. We show herein that these unrelated, nonhomologous peptides clearly compete reciprocally for recognition by the appropriate Kd restricted CTL. In contrast, different NP peptides that are recognized by other CTL restricted by HLA-B37, H-2-Db or KK, either failed to compete or were much less efficient as competitors than NP peptides recognized in the context of Kd. The efficiency of a peptide as a competitor correlated with its potency as an Ag. The most efficient competitor was a variant peptide of NP 147-158 with R156 deleted, which had been previously shown to be 1,000 times more efficient as an Ag than its natural homolog. Our results suggest that peptides recognized by CTL in the context of the same MHC class I restriction element may bind to the same or interdependent site(s) on the restriction molecule.     

Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. Immune response of BALB/c mice to human immunodeficiency virus envelope glycoprotein.

In vivo priming of CTL requires the association with MHC class I molecules of peptides derived from the processing of endogenously produced proteins. Immunization with exogenous proteins or peptides rarely induces MHC class I-restricted CTL unless they are associated with lipidic compounds. The capacity to induce CTL was compared in synthetic peptides and simple lipopeptides containing the Immunodominant MHC class I H-2Dd-restricted T-cell epitope of HIV-1 gp160. In contrast with free peptides in saline, lipopeptides induced strong primary CTL responses in vivo. These CTL were able to lyse cells infected with a recombinant vaccinia virus expressing the HIV-1 env gene. Priming of CTL was also successful when using 16-amino acid lipopeptides as 34-amino acid lipopeptides, suggesting that several epitopes might be included in a single construct. In vivo priming of CTL also requires CD4+ T cell help. We therefore searched for Th cell activation after priming with lipopeptides. Our results show that, as with CTL induction, Th cell activation with lipopeptides did not require mixing with adjuvant. In addition, lipopeptides were also efficient at stimulating antibody-mediated responses. Our results show that a single lipopeptidic construct can induce a total immune response, which is of importance in vaccine development.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells.

A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.     

HIV envelope protein inhibits MHC class I presentation of a cytomegalovirus protective epitope

CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two L(d)-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.     

Identification of residues necessary for clonally specific recognition of a cytotoxic T cell determinant.

The residues in an influenza nucleoprotein (NP) cytotoxic T cell determinant necessary for cytotoxic T cell (CTL) recognition, were identified by assaying the ability of hybrid peptides to sensitize a target cell to lysis. The hybrid peptides were formed by substituting amino acids from one determinant (influenza NP 147-158) for the corresponding residues of a second peptide (HLA CW3 171-182) capable of binding to a common class I protein (H-2Kd). Six amino acids resulted in partial recognition; however, the presence of a seventh improved the potency of the peptide. Five of the six amino acids were shown to be required for recognition. The spacing of the six amino acids was consistent with the peptide adopting a helical conformation when bound. The importance of each amino acid in CTL recognition and binding to the restriction element was investigated further by assaying the ability of peptides containing point substitutions either to sensitize target cells or to compete with the natural NP sequence for recognition by CTL. The T cell response was much more sensitive to substitution than the ability of the peptide to bind the restriction element. Collectively the separate strategies identified an approximate conformation and orientation of the peptide when part of the complex and permitted a potential location in the MHC binding site to be identified. The model provides a rationalization for analogues which have previously been shown to exhibit greater affinity for the class I molecule and suggests that the binding site in major histocompatibility complex (MHC) class I molecules might have greater steric constraints that the corresponding area of class II proteins.     

Induction of class I MHC-restricted, peptide-specific cytolytic T lymphocytes by peptide priming in vivo

The present study investigated the possibility that protein Ag fragments in the form of peptides could serve as the priming Ag in the generation of a MHC class I-restricted immune response. Trypsin-digested chicken ovalbumin (OVA-TD) fragments were used as the model Ag. The results demonstrate the peptides within OVA-TD, when injected into C57BL/6 mice, could prime T cells which lysed H-2b Ia-EL4 target cells in an OVA-TD-specific manner. In contrast to priming with OVA-TD, immunization of mice with intact OVA did not lead to generation of CTL against OVA-TD or OVA. Furthermore, target cells sensitized with intact OVA failed to be recognized by OVA-peptide-specific CTL indicating that the target cells serving as APC were unable to generate the relevant peptide determinants recognized by the T cells. These results support the idea that the processing pathway within APC for class I-restricted T cells may differ from that used for class II-restricted T cells. Using OVA-TD-specific CTL clones (phenotypically Thy 1+, CD8+, CD4-, Pgp-1+) isolated from primed animals to screen OVA-TD fractions separated by HPLC, two T cell peptide determinants were identified corresponding to OVA sequences 111-122 and 370-381. Both determinants were recognized by CTL clones in the context of the H-2Db molecule.     

Specificity of peptide binding by the HLA-A2.1 molecule

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.     

Recognition of a sequestered self peptide by influenza virus-specific CD8+ cytolytic T lymphocytes

The Ag receptors on CD8+ CTL recognize foreign antigenic peptides associated with cell surface MHC class I molecules. Peptides derived from self proteins are also normally presented by MHC class I molecules. Here we report that an H-2Kd-restricted murine CD8+ CTL clone directed to an influenza hemagglutinin epitope can recognize a peptide derived from the murine mitochondrial aconitase enzyme in association with H-2Kd molecules. Surprisingly, this self peptide is not normally displayed on the cell surface associated with the restricting MHC class I molecule. Several lines of evidence suggest that this self peptide, although requiring association with the Kd molecule for CTL recognition, is not associated with this or other MHC class I allele under physiologic conditions in intact cells. Rather, it is sequestered in the cytoplasm associated with a carrier protein and is released only upon cell disruption. These results suggest a means of restricting the entry of self peptide into the class I pathway. In addition, this finding raises the possibility that self peptides sequestered within the cell can, after release from damaged cells, interact with MHC class I molecules on bystander cells and trigger autoimmune injury by virus-specific CTLs during viral infection.