HLA-Independent Heterogeneity of CD8⁺ T Cell Responses to MAGE-3, Melan-A/MART-1, gp100, Tyrosinase, MC1R, and TRP-2 in Vaccine-Treated Melanoma Patients

Patients

We evaluated 22 sequential, vaccine-treated, HLA-A*02⁺ patients with resected malignant melanoma. Seven (32%) had American Joint Committee on Cancer (AJCC) stage II, nine (41%) had stage III and six (27%) had stage IV disease. HLA typing was performed by complement-mediated cytotoxicity. HLA-A2 subtyping was performed by the PCR-SSP (sequence specific primers) technique using the set containing 5′ and 3′ primers for identifying A*0201 to A*0217 alleles (Dynal, Oslo, Norway) (20).

Vaccine and immunization

All patients were immunized to a polyvalent melanoma vaccine prepared, as previously described, from Ags shed into culture medium by a pool of melanoma cell lines (SFM14, SFM20 and SFSKMel28) (21, 22). Briefly, the shed material was collected, concentrated, pooled, treated with 0.5% Nonidet P-40 to break up aggregates, and ultracentrifuged at 100,000 × g for 90 min to deplete alloantigens. The supernatant was filter sterilized, adjusted to a protein concentration of 200 μg/ml, dispensed into sterile glass vials, and frozen at −80°C until used. The vaccine contains MAGE-1, MAGE-3, Melan-A/MART-1, and tyrosinase by Western blotting (14), gp-100 by immune precipitation, and several other melanoma-associated Ags of 45–110 kDa that are expressed by melanoma tissue in vivo and are immunogenic in humans (23). All patients were immunized to vaccine, using alum or liposomes as adjuvant (24). It was administered intradermally split into the four extremities, every 2–3 wk four times. Peripheral blood was collected before immunization and 1 wk following the fourth immunization. Mononuclear cells were separated on Ficoll-hypaque and frozen in liquid N₂ until used.

Preparation of peptides

Peptides were prepared on an Applied Biosystems (Foster City, CA) instrument. Briefly after removal of the α-amino-ter-butylcarbonyl protecting group, the phenylacetamidomethyl resin peptide was coupled with a 4-fold excess of preformed symmetrical anhydride (hydroxybenzyltriazole esters for arginine, histidine, asparagine, and glutamine) for 1 h in dimethylformamide. For arginine, histidine, asparagine, glutamine, and histidine residues, the coupling step was repeated to obtain a high efficiency coupling. Peptides were cleaved by treatment with hydrogen fluoride in the presence of the appropriate scavengers. Synthetic peptides were purified by reverse phase HPLC. The purity and identity of the peptides, which was routinely >95% was determined by amino acid sequence and mass spectrometric analysis respectively.

Assay of peptide binding affinity to HLA-A*0201

Peptide binding to purified HLA-A*0201 molecules was measured as previously described (25). Briefly, the assay is based on the inhibition of binding to detergent-solubilized HLA molecules of a radiolabeled standard peptide with strong binding affinity for HLA-A*0201. The standard peptide, FLPSDYFPSV, was radioiodinated by the chloramine T method using ¹²⁵I (ICN, Irvine, CA). HLA-A*0201 concentration yielding ∼15% of bound peptide (approximately in the 10 nM range) was used in the inhibition assays. Various doses of the test peptides (1 nM to 10 μM) were incubated with 5 nM radiolabeled standard peptide and HLA-A*0201 molecules for 2 days at room temperature in the presence of a mixture of protease inhibitors and 1 μM β₂-microglobulin (Scripps Laboratories, San Diego, CA). At the end of the incubation period, the percent HLA bound radioactivity was determined by gel filtration. The peptides were selected based on their capacity to bind to HLA-A*0201 with an affinity greater than an IC₅₀ of 500 nM, which is known to be in the immunogenic range for cytotoxic T lymphocyte epitopes (26).

Assay of peptide-specific CD8⁺ T cells

We determined the number of peptide-specific CD8⁺ T cells in peripheral blood by filter spot assay as previously described (14). Briefly, 96-well polyvinylidene dilfuoride filter plates (Millipore, Bedford, MA) were precoated with mAbs to human IFN-γ, (Biosource, Camarillo, CA) and seeded with ∼20,000 HLA-A*0201⁺ target cells/well (SFM20 · A2) (14). The target cells were pulsed with 20 nM of test peptide before addition of effectors. The HLA-A*0201-restricted influenza peptide FluM1_58–66 (GILGFVFTL) to which most individuals have a CD8⁺ T cell response was added to some wells as a positive control (27). mAb IVA12 (anti-HLA-DR, DP, and DQ (HB145 from the American Type Culture Collection, Manassas, VA) was added to all wells to prevent presentation of class II Ags by targets or residual monocytes. Effector PBMC were thawed, resuspended in RPMI 1640 with 10% FBS and depleted of monocytes on plastic. The cells were then added to the wells, incubated 48 h at 37°C and 5% CO₂, washed with PBS-Tween 20, incubated overnight with goat anti-IFN-γ (R&D, Minneapolis, MN), followed by biotinylated rabbit anti-goat Ig (Sigma, St. Louis, MO) and extravidin alkaline phosphatase (Sigma). Spots representing individual T cells that had been stimulated by peptides and released IFN-γ were visualized with BCIP/NBT (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and counted with a dissecting microscope.

The number of CD8⁺ T cells was calculated as the number of IFN-γ-secreting cells in wells without Abs minus those in wells with anti-CD8 Abs (OKT8 from the American Type Culture Collection). The number of peptide-specific CD8⁺ T cells was determined by subtracting the number of CD8⁺ cells reacting to targets pulsed with an A*0201-restricted control peptide (YLKKIKNSL from falciparum malaria) (28) from the number of CD8⁺ T cells reacting to melanoma peptide-pulsed targets. All assays were conducted twice, once in duplicate and once in triplicate, and the average value used. The mean SD was ±0.95. The test was considered positive if there were ≥5 peptide-specific CD8⁺ T cells per 500,000 PBL, which is twice the maximum SD. The number of vaccine-induced peptide-specific CD8⁺ T cells was calculated by subtracting the number of peptide-specific CD8⁺ T cells present before treatment from that after four vaccine immunizations in the same patient. The vaccine was considered to have stimulated a CD8⁺ T cell response to that peptide if this number was ≥5.

Selection of peptides

The peptides used in this study were selected for their potential ability to stimulate CD8⁺ T cells in vivo based on being derived from Ags known to be associated with melanoma, i.e., MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, or TRP-2; having 9–10 amino acid residues; containing an HLA-A*0201 binding motif; and by having high binding affinity for HLA A*0201 in vitro. A total of 45 melanoma peptides that satisfied these criteria is provided in Table I⇓.

CD8⁺ T cell responses to individual peptides

We examined the ability of the 45 HLA-A*0201-restricted peptides selected as described above, an HLA-A*0201-restricted malaria peptide as a negative control (28), and an HLA-A*0201-restricted influenza peptide as a positive control (27) to be recognized by CD8⁺ T cells from melanoma patients. We measured the number of CD8⁺ T cells directed to each peptide before and after immunization to a polyvalent melanoma vaccine containing the Ags from which the melanoma peptides were derived. The study was conducted on 22 sequential HLA-A*02⁺ patients who were immunized to the polyvalent vaccine. All but one patient were A*0201⁺. Patient 484 was A*0205+.

Before vaccine treatment, elevated numbers of circulating peptide-specific CD8⁺ T cells were found in 8 of the 22 patients. These patients reacted to 14 of the 45 melanoma peptides (see Table I⇑). One or more of these peptides was derived from each of the 7 Ags studied. However, elevated CD8⁺ T cells to any one peptide were present in no more than 1 patient (5%).

Following four vaccine immunizations, a peptide-specific CD8⁺ T cell response was induced or augmented to 22 (47.8%) of the peptides, as indicated by a minimum increase of 5 peptide-specific CD8⁺ T cells over baseline measurement in the same patient (see Table II⇓). Of the induced responses, at least one of the inducing peptides was derived from each of the proteins studied. The most frequently positive peptides were gp100_585–613 IMPGQEAGL) to which responses were induced in 3 (14%) of the 22 patients (see Table II⇓). The highest vaccine-induced responses were directed to MAGE-3_271–278 (FLWGPRALV), Melan-A/MART-1_56–64 (ALMDKSLHV) and gp100_13–21 (AVIGALLAV) which all were >40 peptide-specific CD8⁺ T cells/500,000 PBL over baseline measurements. Twenty-one of the 22 patients were also tested for their ability to recognize the HLA-A*0201-restricted influenza peptide FluMI_58–66 (27) as a positive control. This peptide was recognized by 76% of the patients. There was no response to the control malaria peptide in any patient either before or after vaccine immunization. Overall, vaccine immunization induced a peptide specific CD8⁺ T cell response to at least one peptide in 59% of the 22 patients. Responses were induced in 7 of 14 (50%) of patients without a pre-existing response, and further induced in 4 of 8 (50%) patients who had a pre-existing response before vaccine treatment.

Relative responses to individual Ags

The relative responses to each Ag were evaluated based on the frequency of vaccine-induced, peptide-specific, CD8⁺ T cell responses to at least one peptide derived from that Ag. The results are summarized in Table III⇓. All of the melanoma Ags stimulated a CD8⁺ T cell response to at least one peptide on that Ag. The most frequently recognized Ag was gp100, which induced CD8⁺ T cell responses in 27% of patients. Overall, 59% of the patients had a vaccine-induced CD8⁺ T cell response to at least one of the Ags studied.

Heterogeneity of CD8⁺ T cell responses to individual Ags and peptides

There was striking heterogeneity in the ability of individual patients to develop vaccine-induced CD8⁺ T cell responses to individual peptides on the same Ag. An example of this heterogeneity in responses to peptides of gp100 is illustrated in Table IV⇓. Individual patients immunized to the same amount of gp100-containing vaccine developed CD8⁺ T cell responses to one gp100 peptide but not to others, whereas the reverse was true of other patients. Overall, 27% patients developed a CD8⁺ T cell response to at least one of the gp100 peptides, but no more than three patients (14%) reacted to the same peptide. There was a similar degree of heterogeneity in vaccine-induced responses to peptides derived from the other Ags (data not shown). Nine to 18% of the patients reacted to at least one peptide from each Ag, but no more than three patients (14%) reacted to the same peptide that Ag.

There was also marked heterogeneity in the responses of individual patients to individual Ags. As illustrated in Table III⇑, some patients developed vaccine-induced CD8⁺ T cell responses to peptides of one Ag but not to those of another Ag, whereas the reverse was true for other patients. Though 59% of patients developed CD8⁺ T cell responses to at least one of seven Ags studied, only 9% to 27% of patients reacted to the same Ag.

Correlation of vaccine-induced CD8⁺ T cell responses with clinical outcome

The clinical status of the patients in the study is shown in Table V⇓. All survived beyond 1 year, and over 75% are still surviving. Of those patients who had a vaccine-induced CD8⁺ T cell response to at least one of the Ags tested, a higher proportion (though not statistically significant) were progression-free after 1 year as compared with those who had no response (54% vs 22%). This positive correlation was unrelated to the stage of disease or the type of adjuvant administered with the vaccine as these two variables were equally distributed in both groups. Most likely a larger study will be needed to show statistically significant differences.