Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.     

Induction of HLA-A2-restricted CTLs to the mucin 1 human breast cancer antigen

HLA-A*0201/Kb transgenic mice were immunized with oxidized mannan-mucin 1 (MUC1) as a fusion protein (containing five repeats of the 20-amino-acid MUC1 VNTR (variable number of tandem repeats) that generated highly active CD8+ CTLs to MUC1 peptides. In a direct CTL assay, the MUC1 peptides could be presented specifically by both the transgenic murine HLA-A*0201/Kb and human HLA-A*0201 molecules. The 9-mer MUC1 peptide sequences (APDTRPA and STAPPAHGV) were presented by HLA-A*0201, although they did not contain L at P2 and L/V at P9, the preferred motifs; as a consequence, the binding was of relatively low affinity when compared with a high affinity-binding HIV peptide (ILKEPVHGV). In addition, when mice were immunized separately with the HLA-A*0201-binding peptides (STAPPAHGV or APDTRPAP-containing peptides-keyhole limpet hemocyanin-mannan), direct lysis of MCF-7 (HLA-A*0201+, MUC1+) also occurred. The findings are of interest for tumor immunotherapy, particularly as the CTLs generated to low affinity-binding peptides were highly active and could specifically lyse an HLA-A*0201+ human breast cancer cell line without further in vitro stimulation. The findings demonstrate that the range of peptides that can generate CTLs is broader than formerly considered.     

Immunization with human papillomavirus type 16 (HPV16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV16-induced tumor cells

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.     

CTL response to Mycobacterium tuberculosis: identification of an immunogenic epitope in the 19-kDa lipoprotein

The successful resolution of infection with Mycobacterium tuberculosis (M.tb) is believed to involve the induction of CTLs that are capable of killing cells harboring this pathogen, although little information is known about the MHC restriction or fine specificity of such CTLs. In this study, we used knowledge of the HLA-A*0201-binding motif and an immunofluorescence-based peptide-binding assay to screen for potential HLA-A*0201-binding epitopes contained in the 19-kDa lipoprotein of M.tb (M.tb19). CD8+ T cells derived from HLA-A*0201+ patients with active tuberculosis (TB) as well as tuberculin skin test-positive individuals who had no history of TB were used as effector cells to determine whether these epitopes are recognized by in vivo-primed CTLs. An in vitro vaccination system using HLA-A*0201+ dendritic cells (DCs) as APCs was used to determine whether these epitopes can sensitize naive CD8+ T cells in vitro, leading to the generation of Ag-specific CTLs. The results show that an HLA-A*0201-binding peptide comprised of residues 88 to 97 of M.tb19 (P88-97) is recognized by circulating CD8+ CTLs from both healthy tuberculin skin test-positive individuals and patients with active TB but not by tuberculin skin test-negative subjects. Moreover, dendritic cells pulsed with this peptide induced class I MHC-restricted CTLs from the T cells of healthy unsensitized persons. Finally, CTL lines that were specific for P88-97 were shown to lyse autologous monocytes that had been infected acutely with the H37Ra strain of M.tb. These results demonstrate that M.tb19 elicits HLA class I-restricted CTLs in vitro and in vivo that recognize endogenously processed Ag. Epitopes of the type identified here may prove useful in the design of an M.tb vaccine.     

Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16

Certain human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies, and the E7 and E6 gene products of HPV type 16 are frequently expressed in these lesions. However, cytolytic T-lymphocyte (CTL)-mediated responses to HPV are rarely detectable in patients with cervical cancer. To examine whether the T-cell response is deficient during the HPV-induced transformation, we produced lines of transgenic (Tg) mice that expressed the E6 and E7 oncogenes in keratinized epithelia. The mice developed severe hypertrophy of all keratinized epithelia, but no malignancies were observed. Although epithelial cells from Tg mice could present at least an E7-encoded CTL epitope (E7 49-57), CTLs from these mice were neither primed to nor made tolerant of this epitope. No quantitative or qualitative differences were seen in the CTL responses of the Tg mice compared to those of their littermates following immunization with the peptide E7 49-57. Immunization of Tg mice with the E7 49-57 peptide protected them against a subcutaneous challenge with tumor cells expressing a transfected E7 gene, yet the skin was unaffected, although the cultured skin epithelial cells from Tg mice expressed E7. Our results suggest that the Tg mice were immunologically ignorant of HPV oncoproteins with respect to a CTL response and that a similar type of ignorance may explain why HPV-associated cervical cancer cells can escape immunological destruction.     

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.     

The effect of epitope variation on the profile of cytotoxic T lymphocyte responses to the HIV envelope glycoprotein

To address the relationship between viral and host factors during HIV infection, we analyzed the effect of viral mutations on T cell responses in seropositive, asymptomatic HLA-A2+ individuals using four envelope (env)-specific peptides with the HLA-A*0201 binding motif. We showed that the natural sequence variation was frequent within epitopes located in the C-terminal region of the env glycoprotein and was largely responsible for a lower env-specific cytotoxic T lymphocyte (CTL) activity in the peptide-stimulated cultures. The highest CTL responses in vitro were induced with conserved epitopes D1 and 4.3 that mapped to the N-terminal region of the env glycoprotein. These peptides exhibited high binding affinity for HLA-A*0201 molecules and stimulated CD8+ T cells of relatively limited TCR Vbeta chain repertoire. Decreased CTL activities to the D1 epitope were observed in the absence of any detectable viral mutation, and were associated with lower proliferative responses and expression of the CD28 antigen. Results of this study demonstrate that the degree of sequence variation within a stimulatory epitope of the viral quasispecies, as well as proliferative potential of the effector cells, are among the factors underlying decreased CTL activity in HIV-infected patients. These experiments also provide evidence that the D1 peptide might be useful for the development of vaccines and immune-based therapy.     

Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-A/MART-1 antigenic peptide

HLA-A*0201 melanoma patients often develop a CTL response to an immunodominant peptide derived from the melanocyte lineage-specific protein Melan-A/MART-1. We have shown previously that the antigenic peptide most often involved is the decapeptide Melan-A(26-35) (EAAGIGILTV). We also observed some clonal diversity in the fine specificity of Melan-A-specific CTL. To substantiate this observation, we have now tested a series of Melan-A(26-35) variant peptides containing single alanine substitutions for binding to HLA-A*0201 and recognition by polyclonal and monoclonal Melan-A-specific CTL. Substitution of several residues with alanine reduced peptide binding activity by > 10-fold. In contrast, substitution of E26 with alanine (AAAGIGILTV) resulted in a 5-fold higher binding activity as well as in stronger stability of the corresponding HLA-A*0201/peptide complexes. Interestingly, the peptide variant AAAGIGILTV was recognized more efficiently than the natural decapeptide by short term cultured, tumor-infiltrated lymph node cell cultures and a number of Melan-A-specific CTL clones derived from different individuals. Moreover, this analysis revealed that the fine specificity of the CTL response to the Melan-A immunodominant epitope is quite diverse at the clonal level. At least three distinct patterns of fine specificity were identified. This diversity appears to reflect the diversity of the TCR repertoire available for this Ag, since similar results were obtained with a panel of Melan-A-specific CTL clones derived from a single melanoma patient. These findings have important implications for the formulation of Melan-A peptide-based vaccines as well as for the monitoring of Melan-A-specific CTL responses in melanoma patients.     

Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy

The exclusiveness of the relationship between peptide and HLA alleles, combined with their extensive polymorphism, emphasizes the need for immunization strategies based on endogenous processing of full length proteins (containing multiple epitopic determinants) for presentation to T cells. This could allow vaccination regardless of the patient's HLA phenotype, assuming that individual molecules can be efficient T cell Ags in association with various HLA alleles. An endogenous system of Ag presentation was developed using dendritic cells infected with recombinant viral vectors expressing the melanoma-associated Ag MART-1/Melan A. CD8+ T cells from melanoma patients were activated in vitro by coincubation with infected dendritic cells and tested for recognition of HLA-A-matched melanoma targets. This allowed the analysis of T cell induction in association with any HLA-A allele of a given patient's phenotype. In this system, MART-1/Melan A could not efficiently immunize in association with HLA-A alleles other than A*0201, including the one residue variant from A*0201: HLA-A*0226. Clonal analysis of MART-1/Melan A-specific CTL confirmed that MART-1/Melan A immunodominance is strongly restricted to the AAGIGILTV/HLA-A*0201 combination. The stringent epitope/allele requirements for MART-1/Melan A/TCR interactions were not associated with limitations in the TCR repertoire. In conclusion, autologous induction of MART-1/Melan A CTL by whole Ag processing and presentation is restricted to a unique allele/ligand combination and is excluded by minimal changes in HLA structure. Thus, whole protein vaccination for small m.w. Ags may provide no further advantage over a peptide-based approach.     

Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.     

Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia

Cytotoxic T lymphocyte (CTL) responses to the human papillomavirus (HPV) type 16 E6 and E7 proteins were measured in 20 women with known HPV and cervical disease status. CTL assays were performed after stimulation with E6 or E7 fusion proteins using autologous B lymphoblastoid cells infected with vaccinia viruses expressing E6 or E7. CTL responses to E6 and E7 were detected in 6 (75%) of 8 and 5 (56%) of 9 HPV-16-positive women without cervical intraepithelial neoplasia (CIN), respectively. Responses to E6 or E7 were each detected in only 2 (29%) of 7 HPV-16-positive women with CIN. Responses to both antigens were found in 63% of women without CIN and 14% of those with CIN. CTL responses to E6 or E7 are more commonly detectable in HPV-16-positive women without CIN than in HPV-16-positive women with CIN, suggesting that CTL response may play a role in disease protection.     

Functional dissociation between local and systemic immune response during anti-melanoma peptide vaccination

Peptide vaccination against tumor Ags can induce powerful systemic CTL responses. However, in the majority of patients, no tumor regression is noted. To study this discrepancy, we analyzed CTL reactivity in a melanoma patient (F001) vaccinated with g209-2M peptide, a single residue variant of gp100(209-217). G209/g209-2M-reactive CTL were identified in post- but not prevaccination PBL. Limiting dilution analysis identified one predominant CTL clone (C1-35), with TCR Vbeta6s2, recognizing g209/HLA-A*0201-expressing targets. Additionally, two autologous melanoma lines (F001TU-3 and -4) and 20 separate tumor-infiltrating lymphocyte cultures were generated from a fine needle aspirate of a metastatic lesion progressing after initial response to vaccination. Both F001TU did not express gp100 and were not recognized by C1-35. Loss of gp100 by F001TU correlated with a marked reduction of gp100 expression in the same metastatic lesion compared with prevaccination. Thus, ineffectiveness of C1-35 and tumor progression could be best explained by loss of target Ag expression. Interestingly, 12 of 20 tumor-infiltrating lymphocyte cultures recognized F001TU, but none demonstrated g209/g209-2M reactivity, suggesting a functional dissociation between systemic and local immune response. This study suggests that vaccination effects must be analyzed in the target tissue, rather than in the systemic circulation alone.     

Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge

The development of prophylactic vaccines against retroviral diseases has been impeded by the lack of obvious immune correlates for protection. Cytotoxic T-lymphocyte (CTL), CD4-lymphocyteS, chemokine and/or antibody responses have all been associated with protection against HIV and AIDS; however, effective and safe vaccination strategies remain elusive. Here we show that vaccination with a minimal ovine CTL peptide epitope identified within gp51 of the retrovirus bovine leukemia virus (BLV), consistently induced peptide-specific CTLs. Only sheep whose CTLs were also capable of recognizing retrovirus-infected cells were fully protected when challenged with BLV. This retrovirus displays limited sequence variation; thus, in the relative absence of confounding CTL escape variants, virus-specific CTLs targeting a single epitope were able to prevent the establishment of a latent retroviral infection.     

Association between HLA-DQB1 alleles and risk for cervical cancer in African-American women

Squamous-cell carcinoma of the cervix and its precursor lesions are associated with human papillomavirus (HPV) infection. Epidemiological studies indicate that HPV infection in itself is not sufficient for cervical-cancer induction, suggesting that other factors contribute to carcinogenesis. We have investigated the potential role of host genetic background as one such factor. We screened a series of squamous-cell carcinomas of the cervix for HLA-class-II DQB1* alleles by the polymerase chain reaction and site-specific oligonucleotide probe hybridization and for HPV type from African-American women using a local, ethnically matched control panel. Statistically significant associations for increase in relative risk for cervical cancer were seen for DQB1*0303 and DQB1*0604. DQB1*0201 and the heterozygote DQB1*0301/*0501 showed a decrease in relative risk for cervical cancer. HPV typing revealed no association between virus type and DQB1 alleles. Our results confirm other studies showing an increase in relative risk for cervical cancer associated with HLA-DQ3 alleles in Caucasians.     

The 2,4-dichlorophenol hydroxylase of Alcaligenes eutrophus JMP134 is a homotetramer

Several epitopes in the human melanoma antigens recognized by HLA-A2-restricted CTLs have a relatively low MHC-binding affinity and as a result may be expressed at very low densities on the cell surface, indicating that these epitopes may not be efficient immunogens. To express these epitopes at higher densities on the surface of antigen-presenting cells and therefore improve their immunogenicity, a DNA construct in which a cDNA fragment encoding the melanoma epitope MART-1(27-35) or gp100(280-288) was inserted between sequences encoding the leader and the HLA-A*0201 protein. Cells transfected with these epitope-HLA fusion constructs were recognized by HLA-A2-restricted melanoma-reactive CTLs specific for the MART-1 or gp100 epitope. In addition, tumor-reactive CTLs could be induced from PBMCs of patients with metastatic melanoma by in vitro stimulation with HMY-C1R B-cell lines expressing the MART-1 or gp100 epitope-HLA-A*0201 fusion protein. These epitope-HLA fusion constructs may be useful for the development of immunotherapies for patients with melanoma.     

Human cytotoxic T-cells suppress the growth of spontaneous melanoma metastases in SCID/hu mice

Mice with severe combined immunodeficiency (scid) provide an excellent model for studying interactions between human tumor cells and effector cells of the immune system. Because these animals lack functional B and T lymphocytes, they can accept human tumor xenografts and transfer of human effector cells. Here, we determined the ability of a human melanoma-specific, cytotoxic T-cell line (CTL) in suppressing the growth of spontaneously metastasizing human melanoma cells M24 met (HLA-A11, A33) in scid mice. This CTL line was highly cytotoxic and restricted by HLA-A11 against M24 met melanoma cells in vitro but poorly cytotoxic when tested against a human melanoma cell line that did not express HLA-A11. In order to evaluate the efficacy of this CTL line against M24 met melanoma cells in vivo, randomized groups of animals were given injections of either RPMI culture medium, interleukin 2 (IL-2), CTLs, or CTLs + IL-2. IL-2, per se, did not significantly reduce tumor metastases; however, injection of melanoma-specific, HLA-A11 restricted CTLs into scid mice, 1 day postexcision of the previously induced primary tumor, markedly reduced the number of metastatic foci in the lung and decreased metastatic involvement in lymph nodes. The combination of these CTLs with IL-2 proved even more effective, since almost all lung metastases were eradicated and metastatic involvement in both axillary and inguinal lymph nodes was substantially reduced. Our results indicate that these human CTLs maintain their ability for specific killing of metastasizing melanoma cells in scid mice. Our data suggest that reconstitution of scid mice with a specific group of effector cells (step-wise scid/hu) may be helpful for in vivo evaluation of potentially useful cancer immunotherapy modalities.     

Whole blood production of thromboxane, prostacyclin and leukotriene B4 after dietary fish oil supplementation in man: effect of vitamin E

Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.     

Generation and characterization of gp100 peptide-specific NK-T cell clones

MHC-restricted cytotoxic T lymphocytes (CTLs) specific for antigens expressed by malignant cells are important components of immune responses against human cancer. Peripheral blood monocytes of HLA-A2+ healthy donors were used to induce dendritic cells (DCs) by granulocyte-macrophage colony-stimulating factor and interleukin-4 and loaded with a gp100 peptide (YLEPGPVTA). By applying these peptide-loaded DCs, a CTL line that displayed high cytotoxic reactivity with peptide-loaded target cells was generated. A total of 11 gp100 peptide-specific CTL clones were generated from this cell line. Several of these CTL clones were studied in detail. Of particular interest was clone CTL-45, which, contrary to the parental cell line, displayed strong NK activity and, by flow-cytometric analysis, revealed a CD3+, TCR BV17, CD8+ and CD56+ phenotype. This clone was strictly peptide-specific and effectively killed a panel of melanoma cells expressing HLA-A2 and gp100. Tumor-specific T cells with this kind of dual function are potentially of great clinical importance as they have a backup mechanism that may go into action when tumor cells escape specific killing by losing their HLA-class I molecules.