Cytotoxic T cell reactivity and HLA-B35 binding of the variant Plasmodium falciparum circumsporozoite protein CD8+ CTL epitope in naturally exposed Kenyan adults

In this study, we have investigated the extent of natural polymorphism in the CD8⁺ cytotoxic T lymphocyte (CTL) determinant (amino acids 368–390) of circumsporozoite (CS) protein of Plasmodium falciparum field isolates from a holoendemic region of Kenya, and determined how this variation affects the CTL reactivities in clinically immune adults and binding specificities to human histocompatibility leukocyte antigen (HLA)-B35. Among the eight variant sequences that were found in this region, four were new and not seen in parasites from other geographical regions. When synthetic peptides corresponding to the eight variants were used to test the presence of CTL response in different donors, a different spectrum of CTL reactivity to these variants was noticed. While CTL from some donors recognized the P1 sequence (the most prevalent type of sequence) but not P8 (another major variant), other donors showed a reverse pattern of reactivity. Although none of the donors was able to recognize all the variants, CTL responses to all the eight variant sequences were found in this population. An octamer peptide with P1 sequence KPKDELDY in this polymorphic determinant was known to bind HLA-B35. When we tested the effect of natural variation in this octamer sequence on HLA-B35 binding, it became evident that SP13 with D → N substitution retained its binding specificity to HLA-B35. On the other hand, the SP12 octamer sequence which had two substitutions did not bind HLA-B35. The most interesting finding was the observation that a D → G substitution at position 374 rescued the binding ability of SP14, which otherwise could not bind to this HLA molecule due to E → Q amino acid substitution at position 372. To our knowledge, this is the first demonstration showing that a natural polymorphism can rescue the binding specificity to an HLA-class I molecule that was lost due to another natural amino acid substitution. Altogether, these results demonstrate that natural polymorphism in the CS protein affects both the CTL reactivity and the ability to bind to HLA-B35.     

Epstein-Barr virus isolates with the major HLA B35.01-restricted cytotoxic T lymphocyte epitope are prevalent in a highly B35.01-positive African population

An influence of cytotoxic T lymphocyte (CTL) response over Epstein-Barr virus (EBV) evolution was first suggested by the finding that virus isolates from highly HLA-A11-positive Oriental populations were specifically mutated in two immunodominant A11-restricted CTL epitopes. Here we turn to a second HLA allele, B35.01 and show that B35.01-restricted CTL responses in Caucasian donors reproducibly map to a single peptide epitope, YPLHEQHGM, representing residues 458–466 of the type 1 EBV nuclear antigen 3 A protein (B95.8 strain). In this case, however, most EBV isolates from a highly B35.01-positive population (in The Gambia) either retained the CTL epitope sequence or carried a mutation (P → S at position 2) which conserved antigenicity; changes leading to reduced antigenicity (Y → N at position 1) were found in only a minority of cases. Furthermore, CTL recognizing the YPLHEQHGM epitope could be reactivated from the blood of some B35.01-positive Gambian donors by in vitro stimulation with the synthetic peptide, indicating that epitope-specific immunity does exist in this population. Possible differences between the A11-based and B35.01-based studies are discussed.     

Combined structural and immunological refinement of HIV-1 HLA-B8-restricted cytotoxic T lymphocyte epitopes

This study demonstrates that use of structural information improves the definition and optimization of cytotoxic T lymphocyte (CTL) epitopes. Epitope optimization usually requires numerous truncated peptides or a reverse immunogenetic approach, where the peptide binding motif is used to predict epitopes. These binding motifs do not reliably predict all peptides which are CTL epitopes. Comparison of 24 peptides eluted from HLA-B8 with 10 HLA-B8-restricted defined CTL epitopes demonstrated that known epitopes varied considerably at anchor positions. We used structural information based on determination of the crystal structure of the HLA-B8-GGKKKYKL complex to reassess previously described CTL epitopes, to predict new epitopes, and to predict the consequences of naturally occurring variation within epitopes. These predictions were confirmed by cytotoxicity and binding assays. Use of combined structural and immunological data more accurately defines the true peptide-binding motif of a restriction element than eluted peptide data allows.     

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.     

Identification of a shared epitope recognized by melanoma-specific, HLA-A3-restricted cytotoxic T lymphocytes

We previously established a melanoma-reactive cytotoxic T lymphocyte (CTL) line that recognizes multiple epitopes in the context of HLA-A3. To increase the number of peptides available for use in a vaccine for the treatment of melanoma, we identified one of these epitopes, SQNFPGSQK, through a combination of epitope reconstitution experiments and mass spectrometry. The SQNFPGSQK peptide was also found to be associated with HLA-A3 on an additional melanoma tumor line, thus indicating that the peptide is not unique to the melanoma tumor line from which it was isolated and thus, unlikely to arise through a mutational event. Although the protein origin of SQNFPGSQK has yet to be established, the shared nature of this epitope and the fact that it elicits a natural immune response indicates that it warrants further study to determine its usefulness as a vaccine component for the treatment of melanoma. The peptide may also be useful as a research tool for evaluating spontaneous anti-tumor immune responses in patients with melanoma.     

The role of the conserved residue in pocket A and the polymorphic residue in pocket E of HLA-B*3501 in presentation of human minor histocompatibility peptides to T cells

We investigated T cell recognition for human minor histocompatibility(hmH) peptides using HLA-B^*3501 restricted, hmH specific cytotoxicT lymphocytes (CTL) clones. These CTL clones killed C1R cellsexpressing HLA-B^*3501 but not C1R cells expressing chlmerlcantigens between HLA-B^*3501 and HLA-^*B5101. They also failedto kill C1R cells expressing HLA-B^*3501 mutants at residue 152(B^*3501-V152E) or at residue 171 (B^*3501-Y171H). The CTL clonefailed to kill C1R cells expressing these mutant molecules loadedwith the hmH peptides isolated from C1R-B^*3501 cells althoughit killed a self-B cell line expressing HLA-B^*3501 loaded withthe specific hmH peptides. The CTL clone also failed to killT2 cells expressing the mutant molecules loaded with the specificpeptides whereas it killed T2 cells expressing HLA-B^*3501 loadedwith the specific peptide. On the other hand, naturally occurringspecific hmH peptides were isolated from purified B^*3501-V152Eand B^*3501-Y171H molecules, indicating that both HLA-B^*3501-V152Eand HLA-B^*3501-Y171H molecules can bind the hmH peptides. Thesefindings indicate that both the conserved residue 171 in pocketA and the polymorphic residue 152 in pocket E are critical inrecognition of the T cells but not binding of the hmH peptides.Furthermore, these results provide the possibility that theTCR recognizes a conformational structure of hmH peptides boundto HLA-B^*3501 molecules.     

A MAGE-3 peptide recognized on HLA-B35 and HLA-A1 by cytolytic T lymphocytes

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Antigenic peptide EVDPIGHLY encoded by MAGE-3 and known to be presented by HLA-A*0101 is currently being used in therapeutic vaccination trials. We report here that a cytolytic T-lymphocyte (CTL) clone, which is restricted by HLA-B*3501, recognizes the same peptide and, importantly, lyses HLA-B*3501 tumor cells expressing MAGE-3. These results infer that the current clinical use of peptide EVDPIGHLY can now be extended to HLA-B*3501 patients.     

A MAGE-A1 peptide presented to cytolytic T lymphocytes by both HLA-B35 and HLA-A1 molecules

Antigens encoded by melanoma antigen (MAGE) genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Antigenic peptide EADPTGHSY encoded by MAGE-A1 and known to be presented by HLA-A1 is currently being used in therapeutic vaccination trials. We report here that a cytotoxic T-lymphocyte (CTL) clone, which is restricted by HLA-B35, recognizes the same peptide and, importantly, lyses HLA-B35 tumor cells expressing MAGE-A1. This peptide can be presented to CTL by both HLA-B*3501 and HLA-B*3503 molecules, which are expressed by approximately 19% of Caucasians. These results infer that the current clinical use of peptide EADPTGHSY can now be extended to HLA-B35 patients.     

A peptide derived from melanocytic protein gp100 and presented by HLA-B35 is recognized by autologous cytolytic T lymphocytes on melanoma cells

A panel of autologous cytolytic T lymphocyte (CTL) clones have been isolated from blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. We previously reported the molecular definition of three distinct antigens recognized by some of these CTL clones. We describe here, the identification of a fourth antigenic peptide expressed by this melanoma line and recognized by a CTL clone restricted by HLA-B*3503. The antigenic peptide, which is nine-amino acid long, has the sequence LPHSSSHWL and is derived from melanocyte differentiation antigen gp100. As HLA-B35 is one of the most frequent HLA-B alleles, being present in 20% of the Caucasian individuals, this peptide may be a good target for peptide-based immunotherapy of melanoma.     

An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV-1 envelope glycoprotein

The immunodominant H-2D^d-restricted cytotoxic T lymphocyte (CTL) response to the HIV-1 gp160 envelope glycoprotein maps to a single determinant in the V3 loop, designated p18. Using a series of peptides synthesized on pins we have determined that the minimal core sequence of this determinant required for CTL recognition comprises 8 amino acids (residues 320-327). However, 9mer and l0mer peptides containing this core sequence were more effective than the 8mer peptide at sensitizing D^d-expressing target cells. To analyze the antigenicity of endogenously synthesized p18, minigenes encoding a 10-amino acid determinant (residues 318-327) and a 67-amino acid peptide (residues 281-348; containing the V3 loop) were expressed using vaccinia virus (Vac) recombinants. Both peptides were as effective as wild-type gpl60 in their ability to sensitize target cells for lysis by gpl60-specific CTL. Immunization of BALB/c mice with Vac recombinants encoding both gp160 peptides elicited gp160-specific CTL. These data demonstrate that both the V3 loop itself and a 10-residue epitope are sufficient to prime CTL in vivo and strongly support the potential use of minigene-encoded CTL epitopes for recombinant vaccines designed to induce protective T cell-mediated immunity against HIV-1.     

Identification of a MAGE-1 peptide recognized by cytolytic T lymphocytes on HLA-B*5701 tumors

"Cancer germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. We report the identification of a new MAGE-1-encoded peptide that is recognized by a cytolytic T-lymphocyte (CTL) clone on human leukocyte antigen (HLA)-B*5701. The sequence of the peptide, corresponding to position 102-112 of the MAGE-1 protein sequence, is ITKKVADLVGF. When tumor cells expressing MAGE-1 were transfected with HLA-B*5701, they were lyzed by the CTL clone, indicating that the peptide is processed in tumor cells and can, therefore, be used as a target for anti-tumoral vaccination.     

The high prevalence of the I27 mutant HBcAg18-27 epitope in Chinese HBV-infected patients and its cross-reactivity with the V27 prototype epitope

HBcAg18-27 (FLPSDFFPSV, V27 epitope) is a dominant HLA-A2-restricted epitope in hepatitis B virus (HBV)-infected patients. So far, the occurrence of the epitope has not been assessed in China, where the prevalence of chronic HBV infection is high. In this report, we sequenced the HBV core gene in 105 Chinese patients with chronic HBV infection. Approximately 93.3% (98/105) of the core genes that were sequenced contained mutations with amino acid substitution at position 27 of the core protein: a mutation from a valine to an isoleucine (V27I). The mutant peptide (FLPSDFFPSI, I27) was found to bind to the HLA-A2 molecule with high affinity and elicit specific cytotoxic T lymphocyte (CTL) responses in acutely infected hepatitis B patients. In CTL assays using I27-specific pentamer staining, the V27 epitope showed a cross-reactive T cell response specific for the I27 epitope, but not vice versa. These findings provide important insights for the design of HBcAg18-27-based vaccines in the future.     

Induction of a cytotoxic T cell response by co-injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund's adjuvant (IFA): Further enhancement by pre-injection of IFA alone

We have previously demonstrated that it is possible to induce a consistent and strong cytolytic T lymphocyte (CTL) response to synthetic peptides, corresponding to poorly immunogenic malaria CTL epitopes, by co-injecting them with peptides representing defined T helper (Th) epitopes in incomplete Freund's adjuvant (IFA). In this study we have tested different immunization protocols to improve further the elicitation of the CTL response. We show that the CTL response to a mixture of Th + CTL peptides administered in IFA was further enhanced by a previous injection of the Th epitope peptide in IFA. Moreover, we found that the response could be significantly augmented by a pre-injection of IFA alone. This enhancement was observed only if the Th epitope was also present in the second injection. The number of lymph node cells recovered was 2–3-fold higher in mice pre-injected with IFA, but the increase in specific CTL activity, expressed as lytic units per animal, by pre-injection of IFA was at least 10–20-fold. Thus, pre-injection of IFA clearly increases the magnitude of a subsequent CTL response.     

Sequence analysis of the novel HLA-B35 (B*3576) allele in an African individual

A novel human leucocyte antigen (HLA)-B35 (HLA-B*3576) allele has been described in an African individual by polymerase chain reaction sequence-based typing. This new allele contains six nucleotide substitutions and is homologous to B*3501 with the exception of residues 66-74 resulting in five amino acid mutations.     

CTL from EIAV carrier horses with diverse MHC class I alleles recognize epitope clusters in Gag matrix and capsid proteins

Cytotoxic T lymphocytes (CTL) are important for controlling equine infectious anemia virus (EIAV). Because Gag matrix (MA) and capsid (CA) are the most frequently recognized proteins, the hypothesis that CTL from EIAV-infected horses with diverse MHC class I alleles recognize epitope clusters (EC) in these proteins was tested. Four EC were identified by CTL from 15 horses and 8 of these horses had diverse MHC class I alleles. Two of the eight had CTL to EC1, six to EC2, five to EC3, and four to EC4. Because EC2-4 were recognized by CTL from >50% of horses with diverse alleles, the hypothesis was accepted. EC1 and EC3 were the most conserved EC and these more conserved broadly recognized EC may be most useful for CTL induction, helping overcome MHC class I polymorphism and antigenic variation.     

Inhibition of tumor growth by peptide specific cytotoxic T lymphocytes in a three-dimensional collagen matrix

Tumor associated, MHC I restricted antigenic peptides have been identified in both human and mouse tumors. Cytotoxic T lymphocytes (CTL) which recognize these tumor associated antigenic peptides are potential anti-cancer effectors. The anti-tumor activity of CTL is usually measured in vitro by the ⁵¹Cr release assay and in mice by tumor growth inhibition which is the most direct assessment of anti-tumor effect. In clinical studies, an in vivo tumor growth inhibition assay is not an option and an in vitro assay which corroborates with in vivo tumor growth is needed to assess the long-term outcome of CTL activity. Here, a three-dimensional (3-D) collagen gel assay was developed to measure in vitro the inhibition of mouse mammary tumor growth by anti-tumor CTL. BALB/c mouse CTL were induced with peptide E474 SFAVATTAL which was expressed by mouse mammary tumor cells D2F2. To measure D2F2 tumor growth inhibition in vitro, a mixture of tumor cells and anti-E474 CTL in a 1 μl cell bolus was embedded in the collagen gel. Complete eradication of tumor growth was observed at E:T ratio of or greater than 1:1. rIL-2 supplementation was necessary to achieve long-term tumor growth inhibition. Even spontaneous D2 tumor explant could be grown in the collagen gel and addition of anti-E474 to this culture reduced tumor growth. This assay system provides a realistic and sensitive alternative to the in vivo tumor growth inhibition assay and allows easy adaptation to test additional therapeutic reagents.     

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes

CD8⁺ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide / TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-γ and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-γ and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be usefol for inducing T lymphocytes with qualitatively different effector functions.     

Silencing of endogenous IL-10 in human dendritic cells leads to the generation of an improved CTL response against human melanoma associated antigenic epitope, MART-1 27-35

Dendritic cells (DC) present antigenic epitopes to and activate T cells. They also polarize the ensuing T cell response to Th1 or Th2 type response, depending on their cytokine production profile. For example, IL-12 producing DC generate Th1 type T cell response whereas IL-10 producing DC is usually tolerogenic. Different strategies--such as the use of cytokines and anti-cytokine antibodies, dominant negative forms of protein, anti-sense RNA etc.--have been employed to influence the cytokine synthetic profile of DC as well as to make DC more immunogenic. Utilizing GFP expressing recombinant adenoviruses in association with lipid-mediated transfection of siRNA, we have silenced the endogenous IL-10 gene in DC. We show that IL-10 gene silenced DC produces more IL-12 and also generates a better cytolytic T cell response against the human melanoma associated epitope, MART-1(27-35), in vitro. We also show that the GFP expressing adenoviral vector can be used to optimize the parameters for siRNA delivery in primary cells and show that RNA interference methodology can efficiently knock down virus encoded genes transcribed at very high multiplicity of infection in DC.