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Immunology |
Department of Immunology and Cancer Center, Mayo Clinic and Mayo Graduate School, Rochester, Minnesota 55905
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several TAAs constitute possible targets for developing T-cell immunotherapy against malignant melanoma (9 , 21) . Of these, the melanocyte-related antigen gp100 is probably the most studied and is currently being evaluated in the clinic (4 , 22) . Numerous CTL epitopes restricted by the MHC class I alleles HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-Cw8 have been reported (22, 23, 24, 25, 26, 27, 28, 29) . On the other hand, only one HTL epitope, which is restricted by the HLA-DR*0401 MHC class II allele expressed in approximately one-fourth of the population, has been described (30 , 31) . Thus, to extend population coverage for melanoma patients, it becomes necessary to identify additional HTL epitopes, preferably of the promiscuous MHC class II type.
In the present study, we used a computer-based algorithm to identify peptide sequences from gp100 with potential promiscuous MHC class II binding characteristics (32) . Three synthetic peptides corresponding to potential promiscuous HTL epitopes were selected, synthesized, and tested for their capacity to stimulate gp100-specific CD4+ T cells in vitro using blood cells from healthy volunteers. The results show that three of the predicted epitopes were able to trigger HTL responses in individuals expressing diverse HLA-DR alleles. The T-cell response to one of these peptides, gp100175–189, was found to be restricted by either the HLA-DR53 or the DQw6 alleles, and the responses to the other peptide epitopes (gp10074–89 and gp100576–590) were restricted by HLA-DR7. In all cases, the positions of the newly identified HTL epitopes in the gp100 sequence were found to lie within or close to previously described CTL epitopes. Some of the therapeutic implications of the physical proximity between HTL and CTL epitopes are addressed in the "Discussion."
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthetic Peptides and Algorithm Analysis.
Potential MHC class II promiscuous helper T-cell epitopes were predicted from the amino acid sequence of the gp100 antigen using the algorithm tables for three HLA-DR alleles (DRB1* 0101, DRB1*0401, and DRB1*0701) published by Southwood et al. (32)
. A computer program based on Microsoft Excel was fashioned for the three HLA-DR alleles. For each position of a 9-amino acid sequence-binding core, a value was assigned for each possible specific amino acid using the tables published by Southwood et al. (32)
. The computer program calculated the ARB values for each possible 9-mer of the gp100 sequence. The rationale for this approach is that the higher the ARB value of a peptide, the higher the probability that the peptide will bind to the corresponding HLA-DR allele. Peptides that displayed high ARB values and were proximal to known CTL epitopes were synthesized and purified as described. The purity (>95%) and identity of peptides were determined by high-performance liquid chromatography and mass spectrometry.
In Vitro Induction of Antigen-specific HTLs Using Synthetic Peptides.
The method used for generating tumor antigen-reactive HTL lines and clones using peptide-stimulated PBMCs has been described in detail previously (13
, 14)
. Briefly, DCs were generated in tissue culture from adherent monocytes that were cultured for 7 days in the presence of GM-CSF and interleukin 4. A total of 1 x 104 peptide-pulsed DCs (10 µg/ml for 2 h) were irradiated (4200 rad) and cocultured with 3 x 104 autologous purified CD4+ T cells (using antibody-coated magnetic beads from Miltenyi Biotech) in each well of a 96-well round-bottomed culture plate. Seven days later, the cultures were restimulated with peptide-pulsed irradiated autologous PBMCs, and 2 days later, human recombinant interleukin 2 was added at a final concentration of 10 IU/ml. After 7 days, the microcultures were tested for their proliferative responses to peptide as described below. Those microwells showing a proliferative response to peptide of at least 2.5-fold over background were expanded in 24- or 48-well plates by weekly restimulation with peptides and irradiated autologous PBMCs. In some instances, T-cell lines were cloned by limiting dilution and used for additional studies. Culture medium for all procedures consisted of RPMI 1640 supplemented with 5% human male AB serum, 0.1 mM MEM, nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 µg/ml gentamicin. The Institutional Review Board on Human Subjects (Mayo Foundation) approved this research, and informed consent for blood donation was obtained from all volunteers.
Antigen-specific Proliferative Response of T Cells.
T cells (3 x 104 cells/well) were mixed with irradiated APCs in the presence of various concentrations of antigen (peptides, tumors, or tumor lysates) in 96-well culture plates. APCs consisted of either PBMCs (1 x 105 cells/well), HLA-DR-expressing L cells (3 x 104 cells/well), or EBV-LCLs or melanoma tumor cells (3 x 104 cells/well) pretreated with IFN-
(500 units/ml for 48 h) to enhance MHC expression. Tumor cell lysates were prepared by three freeze/thaw cycles of 1 x 108 tumor cells resuspended in 1 ml of serum-free RPMI 1640. Lysates were used as a source of antigen at 5 x 105 cell equivalents/ml. Cell proliferation assays were incubated at 37°C in a 5% CO2 incubator for 72 h, and during the last 16 h, each well was pulsed with 0.5 µCi [3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ). In some cases, culture supernatants were collected before the addition of [3H]thymidine for the determination of lymphokine production using ELISA kits (PharMingen, San Diego, CA). The radioactivity incorporated into DNA, which correlates with cell proliferation, was measured in a liquid scintillation counter after harvesting the cell cultures onto glass fiber filters. To identify the MHC restriction molecules involved in antigen presentation, inhibition of the antigen-induced proliferative response was determined by the addition of anti-HLA-DR Mab L243 (IgG2a; prepared from hybridoma supernatants obtained from American Type Culture Collection) or anti-HLA-DQ Mab SPVL3 (IgG2a; Beckman Coulter, Inc., Fullerton, CA). Both antibodies were used at a final concentration of 10 µg/ml throughout the 72-h assay. The specificity of these antibodies and their capacity to specifically inhibit T helper responses have been determined in our laboratory on numerous occasions. All assessments of proliferative responses were carried out at least in triplicate, and results correspond to the means. The stimulation index was calculated by dividing the mean radioactivity (cpm) obtained in the presence of antigen by the mean radioactivity (cpm) obtained in the absence of antigen but in the presence of APCs.
Antigen-specific Cytotoxicity Assays.
The capacity of HTL lines and clones to kill tumor cells expressing the appropriate peptide-MHC complexes was evaluated using the JAM assay (33)
, which measures the DNA retained by target living cells after incubation with effector T cells. Briefly, target cells were labeled with [3H]thymidine for 18 h in tissue culture, and after washing and counting the cells, they were incubated at various E:T ratios in 96-well round-bottomed plates for 36 h. The cultures were then harvested onto glass fiber filters, and the amount of radioactivity retained in DNA was measured in a scintillation counter. The percentage of cytotoxicity was calculated by the ratio of cpm obtained in the absence of T cells:cpm obtained in the presence of different amounts of T cells x 100. All determinations were done in triplicate.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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show the sequences, the ARB values for each allele, and the ranks and rank sum values for the top 10 candidate peptides. When examining the position that these peptides occupy within the gp100 molecule, it became evident that several of these peptides were proximal to or overlapped with known MHC class I-restricted CTL epitopes (Table 1)
. Because it would be attractive to use relatively small peptides (<25 residues) containing both CTL and HTL epitopes as therapeutic vaccines, we decided to focus on those peptide sequences that were proximal to the known CTL epitopes (marked with an asterisk in Table 1
). Another factor that we took into account to narrow down the number of peptide candidates was the potential problem that these peptides could pose regarding their synthesis, purification, solubility, and stability, all which could be important for developing a suitable vaccine. With these parameters in mind, we decided for the time being to eliminate peptides gp100289–304 (which contains a C residue that could form disulfide bonds) and gp100601–615 (which is extremely hydrophobic) from our T-cell epitope analysis. We thus selected three peptides (shown in italics in Table 1
) to evaluate whether they could induce antigen-specific HTL responses after in vitro immunization.
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. In both cases, the T cells responded to peptide presented by autologous PBMCs in a dose-dependent manner (Fig. 1, a and b)
. However, the affinity of T-cell response to peptide gp100576–590 appeared to be higher (
15-fold) than the response to peptide gp10074–89 as determined by the amount of peptide required to achieve 50% of maximal proliferative response. The responses of both T-cell lines were restricted by HLA-DR7 molecules as determined by the capacity of DR7-transfected L cells to present peptide to the T cells and by the capacity of anti-DR antibodies (but not anti-DQ antibodies) to block the T-cell proliferative responses (Fig. 1, c and d)
. T-cell responses to an irrelevant DR7-binding peptide derived from the MAGE-A3 antigen (30)
were not observed (data not shown), indicating that these T-cell lines were highly specific to the gp100 peptides.
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shows that both of the T-cell clones recognized peptide in a dose-dependent manner with similar levels of affinity (Fig. 2, a and b)
. Interestingly, studies performed to determine the HLA restriction elements for these clones indicated that in one case, the response to peptide gp100175–189 was restricted by HLA-DR53 (Fig. 2c)
, and in the other case, the response to peptide gp100175–189 was restricted by HLA-DQw6 (Fig. 2d)
.
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Recognition of Processed Antigen by Peptide-reactive HTLs.
The data presented above demonstrate that all three candidate peptides from gp100 were indeed capable of inducing CD4+ T-cell responses. Nonetheless, it is critical to determine whether these peptides represent true T-cell epitopes that would be relevant for the development of tumor immunotherapy. Thus, it became important to determine whether APCs that naturally capture and process the gp100 antigen (which bears the putative T-cell epitopes) were capable of stimulating the peptide-reactive T cells. It would be more significant to assess whether APCs could capture antigen derived from dead tumor cells (i.e., freeze/thaw cell lysates) expressing gp100 and process this antigen appropriately to stimulate HTLs. In addition, some melanoma tumor cell lines are known to express surface MHC class II molecules, which may present naturally processed peptides to HTLs. The results presented in Fig. 3a
demonstrate that the HLA-DR7-restricted T-cell line (10B1) induced with peptide gp10074–89 was efficient at recognizing tumor cell lysates from a gp100+ melanoma tumor (697mel), but not control tumor lysates (Jurkat), presented by autologous APCs. For these experiments, we prepared the lysates using a DR7- melanoma cell line (697mel) to ensure that the autologous APCs were indeed capturing the gp100 antigen from the lysate to process and present the epitope to the helper T-cells. In addition, in a separate experiment, lysates prepared from gp100- melanoma cells (A375) did not stimulate the response of the T cells (data not shown).
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. These results indicate that the T-cell epitope represented by peptide gp10074–89 is not only processed by APCs from tumor cell lysates but is also expressed on MHC class II molecules on melanoma cells. A similar analysis was performed using the HTL line induced with peptide gp100576–590, but in this case, T-cell responses could not be detected using tumor lysates or intact melanoma cells (data not shown).
Next, we proceeded to determine whether the two HTL lines reactive with peptide gp100175–189 could recognize naturally processed antigen. HTL clone 5D10 (HLA-DR53 restricted) was unable to recognize melanoma cell lysates presented by autologous APCs (data not shown). However, the HTLs were very effective in recognizing intact HLA-DR53+ melanomas but were not effective in recognizing DR53- melanomas or DR53+ lymphoblastoid B cells that do not produce gp100 (M35-EBV) unless they were pulsed with peptide (Fig. 4)
. Most importantly, the recognition of gp100+ melanoma tumor cells by these HTLs was inhibited by anti-HLA-DR antibodies (Fig. 4)
, confirming that this interaction requires the presentation of peptide by MHC class II molecules.
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demonstrate that these HTLs were capable of reacting with antigen derived from melanoma cell lysates presented by autologous APCs. In addition, this HTL clone was also quite effective in recognizing antigen directly on DQw6+ melanoma cells (Fig. 5b)
. Thus, it appears that the T-cell epitope represented by peptide gp100175–189 is expressed on both HLA-DR53 and HLA-DQw6 molecules on MHC class II+ melanomas.
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, the HLA-DQw6-restricted HTL clone was very efficient in killing the 1102mel cells (DQw6+) but was not efficient in killing a DQw6+ EBV-LCL line that does not express the gp100 antigen. With respect to the other gp100-reactive HTL lines, lower levels of cytotoxicity were observed, but they did not appear to be statistically significant (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recognition of naturally processed antigen by peptide-reactive T cells is an important parameter to determine whether a predicted T-cell epitope is indeed present on tumor cells or APCs, which in our view is critical for determining whether the peptide epitope will be useful for immunotherapy. HTLs may recognize peptide-MHC class II complexes directly on tumor cells that are MHC class II+, and as a consequence, the T cells will become activated and produce cytokines that provide help to CTLs or cytokines that may inhibit tumor cell growth. In some cases, HTLs exhibit cytotoxicity toward antigenic MHC class II tumors (11
, 12
, 17
, 18
, 31
, 34)
. Our results show that HLA-DQw6-restricted HTLs that react with peptide gp100175–189 displayed high levels of cytotoxicity against a DQw6+ melanoma cell line (1102mel; Fig. 6
). However, cytotoxicity was not observed toward another DQw6+ tumor cell line (697mel; data not shown), suggesting that various tumor cell lines may display different levels of susceptibility to lysis by the T cells. The cytotoxic activity of this HTL toward the 1102mel line was not apparent using standard 4-h 51Cr release assays and required the lengthier JAM assay (33)
, suggesting that the cytolytic activity of these cells is different rather than the same as conventional CD8+ CTLs. Most importantly, it remains to be determined whether antitumor cytotoxicity by MHC class II-restricted T lymphocytes takes place in vivo and whether it results in tumor responses.
Tumor antigen-reactive HTLs will also recognize peptide-MHC complexed on APCs that have captured and processed tumor antigen, apoptotic cells, or tumor cell debris (lysates). Our results show that HTLs reactive to two of the three predicted peptide epitopes (gp100175–189 and gp10074–89) were also capable of recognizing naturally processed antigen in MHC class II+ melanomas or in APCs fed with tumor lysates. On the other hand, HTLs that were induced using peptide gp100576–590 failed to proliferate or produce lymphokines when interacting with MHC class II+ melanomas or tumor lysates presented by APCs. The capacity of a HTL to become activated (i.e., proliferate and produce lymphokines) when challenged with APCs that have naturally processed the antigen will depend on two major factors. First, and most important, the peptide epitope must be processed adequately to produce the stimulatory peptide-MHC complexes on the surface of the APCs. It is likely that not all cells have the same capacity to process antigens into MHC class II-binding peptides. For example, a melanoma HLA-DR13-restricted MAGE-A3 HTL epitope was produced by APCs pulsed with either protein or tumor lysates but was not detected on DR13+ MAGE-A3+ melanoma cells, suggesting that this epitope could not be produced via the endogenous pathway (10)
. Our results indicate the absence of responses to APCs pulsed with tumor lysates, but direct recognition on melanoma by the DR53-restricted HTL clone 5D10, specific for peptide gp100175–189 (Fig. 4
; data not shown), suggests that this epitope may not be effectively processed by APCs. However, the same peptide epitope (gp100175–189) was effectively presented by APCs to the HLA-DQw6-restricted HTL clone (Fig. 5a)
. Major differences in affinities of the binding of peptide gp100175–189 to DR53 or DQw6 or differences in the avidities of the HTL clones to their respective peptide-MHC complexes could account for these conflicting results. However, the data presented in Fig. 2, a and b
, suggest that this may not be the case because the peptide titration curves, which assess both the affinity of the peptide to MHC class II and the avidity of the HTLs, can almost be superimposed. One possible explanation for the lack of recognition of tumor lysate-pulsed APCs by the DR53-restricted HTLs could be that antigen processing by these cells does not necessarily result in the exact production of peptide gp100175–189 but may result in the production of a close variant such as gp100176–189 or gp100175–188 that could bind to DQw6 but not to DR53. Studies are under way to determine whether residue truncations at either the NH2- or COOH-terminal ends of peptide gp100175–189 have the same effect or a different effect on the recognition of the epitope by the DR53- and DQw6-restricted HTL clones.
The second parameter that is likely to affect the recognition of processed antigen is the overall affinity (avidity) of the HTL for its peptide epitope. The factors that influence the avidity of T-cell-APC interaction are multiple and rather complex. Most important perhaps are the intrinsic affinity of the T-cell receptor for the peptide-MHC complex and the amounts (or density) of specific peptide-MHC complexes expressed on the APCs. Other factors that may influence the avidity of T cells for their APCs are the presence of adhesion/costimulatory molecules that help stabilize cellular interactions or may even lower the threshold for T-cell receptor activation. Peptide dose responses can provide a rough estimate of the avidity of T cells for their antigen. However, results obtained using peptide titrations can also be affected by the stability of the peptide-MHC complexes on the surface of the APCs. Surprisingly, our results suggest that the HTLs that recognized naturally processed antigen displayed significantly lower avidity (10-fold) for their peptides (gp100175–189 and gp10074–89) than the HTLs (specific for peptide gp100576–590) that failed to respond to natural antigen. In general, we have observed that tumor-reactive HTLs have an intermediate avidity level for their peptides. These HTLs require a concentration of approximately 1 µM peptide to achieve MR50. In contrast, HTLs directed to foreign antigens (viral and bacterial) have a much higher affinity for their ligands, requiring approximately 10–100 times less peptide to reach their MR50 (14
, 35)
. The most likely explanation for the lower avidity of tumor-reactive T cells is that the majority of TAAs are expressed to some extent in normal cells, and as a result, high affinity HTLs may be deleted or tolerized. In accordance with this assumption, a tyrosinase-specific DR4-restricted HTL isolated from a melanoma patient required >10 µM peptide to achieve its MR50 (19)
. However, there are also antitumor HTLs that may have relatively high avidity for their epitopes, as reported by Zarour et al. (18)
, who found that a MART-1-reactive HTL required 0.1 µM peptide to attain its MR50. Thus, our observations that peptide gp100576–590 induced very high avidity HTLs (MR50 0.02 µM; Fig. 2b
) and that these cells were incapable of recognizing naturally processed antigen suggest that this epitope may constitute a cryptic epitope and may not be produced as such by the APCs that process the gp100 protein. Alternatively, we cannot eliminate the possibility that a minute contaminant in our peptide preparation (peptide with unprotected residues) may be highly immunogenic.
Lastly, another parameter that we consider important for the selection of HTL epitopes to be used for improving CTL vaccines is the possibility of using a single peptide of relatively small size (<25 residues) containing epitopes for both CTLs and HTLs. Although this approach may also be achieved by simply linking a CTL epitope with a T helper epitope, we believe that using the antigen’s natural sequence may have significant advantages. First, it is evident that peptides containing more than one T-cell epitope will have to be processed adequately by APCs to produce the separate distinct entities that need to bind effectively to the MHC molecules. Chimeric peptides that are formed by linking two (or more) epitopes found on distant parts of a protein may not be processed adequately to form the exact MHC-binding peptides because the natural protease cleavage sites may have been modified. Furthermore, introducing polylinkers such as AAA or KKK sequences between the epitopes may not necessarily overcome this problem because this also modifies the natural protease cleavage sites. The second major advantage of using peptides of natural sequences containing multiple T-cell epitopes over the use of their linked counterparts is that the latter may contain artificially created sequences that could produce new and potent but irrelevant epitopes that would not be found in the tumor antigen. For example, the new peptide sequence formed from the fusion of CTL epitope with the HTL epitope could constitute a high affinity MHC-binding peptide that may elicit a strong T-cell response, which would not be beneficial but would in fact could be detrimental to the generation of antitumor immunity. Because some degree of peripheral immune tolerance exists to most TAAs, the new artificial epitopes have the potential of being more immunogenic than those corresponding to the TAAs, which could result in the inhibition of the induction of antitumor T cells.
Taking into account the above-mentioned criteria, we focused on potential HTL candidates that were situated in proximity to known CTL epitopes. Notably, peptide gp100175–189, which generated DR53- and DQw6-restricted HTLs capable of recognizing naturally processed antigen, includes a potent HLA-A2-restricted CTL epitope (gp100177–186). Previous studies by our laboratory indicated that peptide gp100177–186 is very effective in inducing antitumor CTL responses by in vitro immunization of PBMCs from normal individuals using peptide-pulsed DCs (24) . Our results show that peptide gp10074–89, which stimulated a HLA-DR7-restricted HTL response, partially overlaps (in its COOH-terminal end) with another CTL epitope, but this epitope is restricted by the HLA-A3 and HLA-A11 alleles (gp10087–95; ALNFPGSQK; Ref. 25 ). Thus, a synthetic peptide of 22 residues (gp10074–95) could be capable of eliciting antitumor CTL and HTL responses in patients expressing HLA-A3 (or HLA-A11) and HLA-DR7. Interestingly, the same HTL epitope (gp10074–89), also overlaps in its NH2-terminal end with another CTL epitope (gp10070–78), which is restricted by HLA-Cw8 (29) .
In summary, we report here the identification two novel HTL epitopes for the melanoma-associated antigen gp100 that are restricted by MHC class II alleles expressed in a large proportion of the population. These epitopes lie proximal to or overlap with previously characterized tumor-reactive CTL epitopes. The present findings suggest that synthetic peptides of relatively small size could be used as therapeutic vaccines that would effectively stimulate both HTL and CTL antitumor responses in melanoma patients. The approach of using peptide vaccines containing both CTL and HTL epitopes was recently described in the HER2/neu antigen system in breast and ovarian cancer patients (36) . These vaccines were shown to increase the CTL precursor frequency to the HER2/neu epitopes contained in these peptides that lasted for more than a year after vaccination, indicating that antigen-specific HTLs may prolong CTL responses.
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FOOTNOTES |
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1 Supported by NIH Grants R01CA80782, R01CA82677, and RR-00585. 
2 To whom requests for reprints should be addressed, at Department of Immunology, GU421A, Mayo Clinic, Rochester, MN 55905. Phone: (505) 284-0124; Fax: (505) 266-5255; E-mail: celis.esteban{at}mayo.edu
3 The abbreviations used are: HTL, helper T lymphocyte; TAA, tumor-associated antigen; PBMC, peripheral blood mononuclear cell; DC, dendritic cell; APC, antigen-presenting cell; MR50, 50% maximal response; EBV-LCL, EBV-transformed lymphoblastoid cell; ARB, average relative binding; GM-CSF, granulocyte macrophage colony-stimulating factor; Mab, monoclonal antibody.
Received 4/12/01. Accepted 8/13/01.
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H. Kobayashi, R. Omiya, M. Ruiz, E. Huarte, P. Sarobe, J. J. Lasarte, M. Herraiz, B. Sangro, J. Prieto, F. Borras-Cuesta, et al. Identification of an Antigenic Epitope for Helper T Lymphocytes from Carcinoembryonic Antigen Clin. Cancer Res., October 1, 2002; 8(10): 3219 - 3225. [Abstract] [Full Text] [PDF]
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R. Omiya, C. Buteau, H. Kobayashi, C. V. Paya, and E. Celis Inhibition of EBV-Induced Lymphoproliferation by CD4+ T Cells Specific for an MHC Class II Promiscuous Epitope J. Immunol., August 15, 2002; 169(4): 2172 - 2179. [Abstract] [Full Text] [PDF]
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G. Zeng, Y. Li, M. El-Gamil, J. Sidney, A. Sette, R.-f. Wang, S. A. Rosenberg, and P. F. Robbins Generation of NY-ESO-1-specific CD4+ and CD8+ T Cells by a Single Peptide with Dual MHC Class I and Class II Specificities: A New Strategy for Vaccine Design Cancer Res., July 1, 2002; 62(13): 3630 - 3635. [Abstract] [Full Text] [PDF]
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 C. Buteau, S. N. Markovic, and E. Celis Challenges in the Development of Effective Peptide Vaccines for Cancer Mayo Clin. Proc., April 1, 2002; 77(4): 339 - 349. [Abstract] [PDF]
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DR) but not by anti-HLA-DQ (
) and M35 EBV-LCL (•).