Although in vitro sensitization assays have shown increased melanoma Ag (MA)-specific CTL reactivity after vaccination with MA peptides, clinical responses have been uncommon. This paradox questions whether data obtained from the in vitro stimulation and expansion of T cells lead to an overestimation of the immune response to vaccines. Using HLA/peptide tetramer (tHLA), we enumerated MA-specific T cell precursor frequency (TCPF) directly in PBMC from 23 melanoma patients vaccinated with gp100:209–217(210M) (g209–2M) peptide. Vaccine-specific TCPF was higher in postvaccination PBMC from seven of seven patients treated with peptide alone and four of five patients treated with peptide plus IL-12 (range of postvaccination TCPF, 0.2–2.4% and 0.2–2.5%, respectively). The increased TCPF correlated with enhanced susceptibility to in vitro stimulation with the relevant epitope. Paradoxically, no increase in postvaccination TCPF was observed in most patients who had been concomitantly treated with IL-2 (1 of 11 patients; range of postvaccination TCPF, 0.02–1.0%), a combination associated with enhanced rates of tumor regression. The lack of increase in TCPF seen in these patients corresponded to inability to elicit expansion of vaccine-specific T cells in culture. This study shows that a peptide-based vaccine can effectively generate a quantifiable T cell-specific immune response in the PBMC of cancer patients, though such a response does not associate with a clinically evident regression of metastatic melanoma.

The identification of melanoma Ags (MA)2 recognized by T cells and their respective HLA class I-restricted epitopes stimulated peptide-based vaccination efforts as a new approach to tumor therapy (1, 2). Although comparative ex vivo sensitization of pre- and postvaccination PBMC has identified reproducible, vaccine-specific systemic T cell responses to immunization, in the majority of cases no regression of tumor is seen (2, 3, 4). We have previously published the results of a vaccination protocol in which patients with metastatic melanoma were sensitized against the gp100 MA by administration of the modified gp100 epitope gp100:209–217(210M) (g209–2M) that is presented in association with the HLA-A*0201 allele. The modification increases ligand affinity to HLA-A*0201/β2-microglobulin complexes and is associated with higher immunogenicity in vitro (5) and in vivo (4) compared with the native epitope. Based on in vitro sensitization assays, 91% of patients that had been injected s.c. with the g209–2M peptide emulsified in IFA demonstrated successful immunization. However, no clinical responses were observed while 13 of 31 patients (42%) treated with the g209–2M peptide plus the systemic administration of high-dose IL-2 demonstrated objective cancer responses (2). Interestingly, only 16% of patients in this second cohort developed immune reactivity. It was suggested that the decreased precursor cell frequency observed when IL-2 was administered could have been due to activation of the vaccine-specific T cells and trafficking to the tumor site. Alternatively, differential susceptibility of vaccine-specific T cells to in vitro sensitization could have been responsible for the discrepant results in the two cohorts of patients rather than different frequency of vaccine-specific T cells in PBMC.

Therefore, the paradoxical behavior of vaccine-elicited immune responses questions, among other variables, the validity of data obtained by ex vivo stimulation and expansion of T cells. These assays require repeated stimulation with arbitrarily chosen concentrations of exogenous epitopes and cytokines, which could alter the functional and phenotypic characteristics of the T cells. Therefore, other assays directly measuring T cell reactivity in PBMC have been fashioned. Among them, the enzyme-linked immunospot (ELISPOT) assay can detect MA-specific CTL directly in PBMC (6) but may overlook inactive epitope-specific T cells that are unable to respond to epitope-specific stimulation. Tetrameric HLA/peptide complexes (tHLA) allow for direct measurement of epitope-specific T cell precursor frequency (TCPF) without in vitro manipulation (7). This method has been successfully used to measure T cell responses to viral infections (8, 9) and has been shown to estimate higher TCPF in PBMC than other assays (10). Furthermore, as this method does not rely on functional T cell responses, supplementary information to functional assays can be obtained.

We analyzed g209- and g209–2M-specific TCPF in pre- and posttreatment PBMC from 23 melanoma patients vaccinated with g209–2M peptide emulsified in IFA. We also evaluated whether enhancement in TCPF secondary to vaccination corresponded to increased susceptibility to in vitro stimulation with the relevant epitope. Furthermore, as the frequency of MA-specific T cells at tumor site might be of greater relevance than in PBMC, we compared, when available, g209-specific TCPF in PBMC and tumor infiltrating lymphocyte (TIL) pairs simultaneously obtained after vaccination.

HLA-A*0201 patients received the g209–2M peptide in IFA. Representative PBMC were obtained from patients treated with peptide in IFA (n = 7, P1–P7), peptide with IL-12 s.c. (IL-12, n = 5, P8–P12), or with high dose (720,000 IU/kg every 8 h) IL-2 i.v. (IL-2, n = 11, P13-P23). These PBMC were selected according to previous in vitro sensitization, suggesting different vaccination outcomes in relation to concomitant cytokine treatment (2). Postvaccination PBMC from the patients treated with peptide alone or peptide with IL-12 had demonstrated tumor specificity after in vitro expansion, whereas PBMC from patients who had been treated with peptide and IL-2 did not. Vaccinations were administered at 3-wk intervals, and blood samples were obtained three weeks after vaccination unless otherwise specified. The HLA class I phenotype of patients was determined on PBMC using sequence specific primer-PCR (11). PCR was also used for molecular subtyping of HLA-A2 (12).

Samples were obtained from blood draws and leukapheresis of melanoma patients before and after vaccination with g209–2M peptide. PBMC were isolated by Ficoll gradient separation and frozen until analysis. For analysis of TCPF in TIL, excised tumor samples were enzymatically digested and frozen without separation of mononuclear cells from tumor cells as previously described (13). Analysis of MA-specific T cells was performed after overnight resting of thawed PBMC in complete medium consisting of RPMI 1640 medium (Biofluids, Rockville, MD) supplemented with 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids), 10 μg/ml ciprofloxacin (Bayer, West Haven, CT), 0.03% l-glutamine (Biofluids), 0.5 mg/ml amphotericin B (Biofluids), 10% heat-inactivated human AB serum (Gemini Bioproducts, Calabasas, CA), and 300 IU IL-2/ml. This procedure allowed depletion of adherent monocytes. PBMC were also analyzed after 10 days of in vitro culture following stimulation with exogenous peptide. This was achieved by the administration of 1 μM peptide in complete medium to the PBMC at the time of thaw and the addition of IL-2 (300 IU/ml) the following day and every third day thereafter.

Tetrameric peptide-HLA-A*0201 complexes were produced as described previously (7). Recombinant HLA-A*0201 heavy chain containing a biotinylation site and recombinant β2-microglobulin were synthesized and used for refolding of soluble HLA (sHLA) molecules in the presence of a HLA-A*0201 binding peptide. sHLA molecules were prepared for the following epitopes: gp100:209–217 (ITDQVTCPFSV, g209); gp100:209–217 (210M) (IMDQVTCPFSV, g209–2M); and FluM1:58–66 (GILGFVFTL, Flu). All peptides were commercially synthesized and purified by gel filtration (Princeton Biomolecules, Columbus, OH). The refolding reaction was dialyzed and concentrated for purification of correctly refolded sHLA on gel filtration. Monomeric sHLA was biotinylated with BirA (Avidity, Denver, CO) at the heavy chain and separated from free biotin by gel filtration. Biotinylated sHLA was tetramerized by adding avidin-PE (Pierce, Rockford, IL) at a 4:1 molar ratio. The final concentration of tetramer was adjusted to 2 μg/ml for g209 and g209–2M tHLA, and to 1 μg/ml for Flu tHLA. As examined by gel filtration, all tHLA were without detectable free avidin-PE.

After overnight depletion of monocytes, nonadherent PBMC were resuspended at 1 × 106 cells/50 μl ice-cold FACS buffer (phosphate buffer plus 5% inactivated FCS; Biofluids) and cells from day 10 CTL cultures were washed and resuspended at 2 × 105 cells/50 μl cold FACS buffer. Cells were incubated on ice with 1 μg tHLA for 15 min, and then continued for 30 min with 10 μl anti-CD8 mAb (100 μg/ml; Becton Dickinson, San Jose, CA). Cells were washed twice in 2-ml cold FACS buffer before analysis by FACS (Becton Dickinson). Fifty thousand events were acquired for CTL cultures and 200,000 events for PBL samples. Staining of tumor preparations was performed similarly to the preparation of PBMC with overnight resting in complete medium and direct staining of nonadherent cells.

Nonadherent PBMC (1 × 106 cells) were stimulated for 6 h with peptide (1 μg/ml) pulsed T2 cells (1 × 106 cell/ml). After 2 h, Brefeldin A (10 μg/ml) (Sigma, Deisenhofen, Germany) was added. After four additional hours, the cells were treated with 4500 U DNase I (Calbiochem, La Jolla, CA) for 5 min at 37°C. EDTA (0.1 M) was added to each well before washing with cold PBS. Cells were fixed with 4% paraformaldehyde for 5 min and washed in PBS containing 0.1% BSA. Cells were blocked overnight with PBS/5% milk on ice in cold room. Permeabilization of cells was performed with PBS. After staining with mAb for 30 min on ice, cells were washed in PBS. Staining with tetramers was performed before fixation of cells. The rest of the procedure was conducted according to the same protocol as all other stains. All samples were analyzed on a Becton Dickinson FACSCalibur flow cytometer using the CellQuest software. Live-gating on lymphocytes, CD3+, and CD8+ was performed during acquisition. The following mAbs were used: allophyocyanin-conjugated mouse anti-human CD3 (IgG1), peridinin-chlorophyll protein-conjugated mouse anti-human CD8 (IgG1), and fluorescein (FITC)-conjugated mouse anti-human CD45RA were purchased from Becton Dickinson (Heidelberg, Germany), and fluorescein (FITC)-conjugated mouse anti-human IFN-γ (IgG1) and fluorescein (FITC)-conjugated mouse anti-human CD45RO (PharMingen, San Diego, CA).

In the light scatter, the lymphocyte population was gated in for evaluation. The frequency (f) of peptide-specific T cells per 106 CD8+ cells was calculated using the following formula: f = URQ/(URQ + LRQ) × 106 CD8+ cells, with URQ containing the tHLA+, CD8+ cells and LRQ containing all other CD8+ cells. From these frequencies, the background with CD8+ staining only was subtracted for each sample to obtain the corrected frequency (fc). The fc is presented as the number of peptide-specific T cells per 1 × 106 CD8+ T cells.

For statistical comparison, the basic unit of analysis was the log10 of change between posttreatment vs pretreatment fc for each day, staining, and stimulation condition. The log10 was chosen because of the wide range of fc values observed in different patients. Because of varying intervals between pre- and posttreatment samples, Spearman correlation analyses and scatter plots were constructed to determine whether there was a relationship between interval (days) from pretreatment to posttreatment and log10 change in fc for each experimental condition.

For each condition, the statistical significance of the log10 change from pre- to posttreatment fc was determined by the Wilcoxon signed rank test, separately for each treatment group. In addition, specific analyses comparing changes under one experimental condition to changes with another condition were also done by the Wilcoxon signed rank test after subtracting one change [log10 (postpre)] from the other.

Finally, the Kruskal-Wallis (KW) test was used to determine the significance of the difference among the three treatment groups with respect to any of the changes or comparisons of the changes from pre- to posttreatment within a particular condition. These p values are indicated as KW in the figures. All p values are two sided and have not been explicitly adjusted for multiple comparisons because all analyses are being done on small groups of patients and with exploratory intent; thus, the results should be interpreted as hypothesis generating until confirmed by other studies.

The TCPF of g209- and g209–2M-specific CD8+ cells were determined in PBMC by g209 and g209–2M tHLA staining as shown in the patient’s sample illustrated in Fig. 1. The precursor frequencies calculated (fc) were then calculated as shown in Table I. Detection of Flu-specific CD8+ cells was included as a control. Because of varying intervals, ranging from 40 to 283 days, between pre- and posttreatment PBMC samples, the effect of the time was assessed by Spearman correlation analysis. The results of the correlation analysis suggested that slight variations in fc could be considered a negligible factor in the interpretation of the results (data not shown). The range of fc noted was from undetectable above background, particularly in the peptide + IL-2 group to 14,806/106 CD8+ cells for g209 (P4, postvaccination), 24,576/106 CD8+ cells for g209–2M (P12, postvaccination), and 21,583/106 CD8+ cells for Flu (P12, prevaccination). The variability of the results and the details for patients in the various cohorts are shown in Table I. Statistical comparison of the pre- and postvaccination fc for each patient in the peptide alone (P alone) treatment group showed a significant increase of g209- and g209–2M-specific CD8+ cells (g209: mean log10 difference (Δ) = 0.73, p = 0.016; g209–2M: Δ = 0.61, p = 0.016) (Figs. 2,A and 3A). A similar change was observed in the peptide + IL-12 (P + IL-12) treatment group (g209: Δ = 0.60, p = 0.125; g209–2M: Δ = 0.78, p = 0.0625). The peptide + IL-2 group (P + IL-2) did not show any obvious trends (g209: Δ = −0.07, p = 0.240; g209–2M: Δ = −0.19, p = 0.17). The differences of trends among various groups were statistically significant (g209: KW = 0.018; g209–2M: KW = 0.0024). Flu fc values were not significantly different between pre- and postvaccination PBMC samples in all treatment groups (P alone: Δ = −0.22, p = 0.30; P + IL-12: Δ = 0.07, p = 0.44; P + IL-2: Δ = −0.58, p = 0.28) (Fig. 4 A). Thus, vaccination-dependent enhancement of TCPF could be detected in peptide treated patients who had not received i.v. IL-2. Sample collection in this study was dictated by the clinical protocol structure, which provided for patient return to our institution for re-evaluation and vaccine administration at 3-wk intervals. However, to investigate whether the relatively low TCPF observed were due to the distance between the last vaccination and sample collection, we tested patients’ PBMC at shorter intervals after vaccine administration. Samples were collected before and 3, 7, 10, and 14 days after vaccination. At no time point were TCPF fluctuations noted (data not shown). Thus, it is unlikely that significantly higher frequencies were missed in the cohort of patients studied because of the delay in which the blood samples had been collected.

FIGURE 1.

Detection of vaccine-specific T cells in PBMC. A patient (P4) who had received two vaccinations with g209–2M peptide demonstrated marked in vivo expansion of g209/g209–2M-specific CD8+ cells in the postvaccination PBMC. Numbers in the URQ indicate the percentage of tHLA staining CD8+ T cells calculated according to the formula: URQ/(URQ + LRQ) × 100.

FIGURE 1.

Detection of vaccine-specific T cells in PBMC. A patient (P4) who had received two vaccinations with g209–2M peptide demonstrated marked in vivo expansion of g209/g209–2M-specific CD8+ cells in the postvaccination PBMC. Numbers in the URQ indicate the percentage of tHLA staining CD8+ T cells calculated according to the formula: URQ/(URQ + LRQ) × 100.

Close modal
Table I.

Calculated precursor frequenciesa of peptide-specific CD8+ cells in PBMC on day 1 (peptide-specific/10e6 CD8+ cells)

g209bg209-2MFluDaysc
PrePostPrePostPrePost
Peptide aloned P1 356 2,109 638 9,664 357 2,254 41 
 P2 667 2,863 2,218 4,916 2,177 3,064 41 
 P3 2,083 3,451 1,558 4,301 1,115 1,274 223 
 P4 1,854 14,806 2,587 23,952 2,870 1e 43 
 P5 104 4,402 1,416 3,440 4,732 5,703 46 
 P6 4,779 9,137 5,473 12,723 6,820 7,901 94 
 P7 454 1,988 408 1,564 415 2,389 41 
         
Peptide+ IL-12 P8 508 4,177 500 5,609 2,002 1,237 42 
 P9 497 2,199 401 1,714 480 667 90 
 P10 282 3,135 243 3,488 836 1,386 40 
 P11 8,566 6,834 12,490 13,618 2,054 4,164 42 
 P12 4,770 14,549 2,274 24,576 21,583 16,650 42 
         
Peptide+ IL-2 P13 2,141 121 1,735 853 1,992 283 
 P14 1,895 1,714 1,607 2,335 663 1,026 199 
 P15 1,842 9,524 1,791 10,043 1,519 4,941 89 
 P16 7,225 4,887 4,620 2,771 2,228 1,473 184 
 P17 433 300 533 14,496 6,309 45 
 P18 2,345 971 1,755 272 421 145 
 P19 616 271 1,051 251 1,690 1,656 54 
 P20 2,558 950 769 979 908 2,687 50 
 P21 1,870 239 895 306 838 260 94 
 P22 3,134 1,813 4,507 1,338 1,838 1,341 41 
 P23 2,427 1,057 2,426 869 1,374 465 85 
g209bg209-2MFluDaysc
PrePostPrePostPrePost
Peptide aloned P1 356 2,109 638 9,664 357 2,254 41 
 P2 667 2,863 2,218 4,916 2,177 3,064 41 
 P3 2,083 3,451 1,558 4,301 1,115 1,274 223 
 P4 1,854 14,806 2,587 23,952 2,870 1e 43 
 P5 104 4,402 1,416 3,440 4,732 5,703 46 
 P6 4,779 9,137 5,473 12,723 6,820 7,901 94 
 P7 454 1,988 408 1,564 415 2,389 41 
         
Peptide+ IL-12 P8 508 4,177 500 5,609 2,002 1,237 42 
 P9 497 2,199 401 1,714 480 667 90 
 P10 282 3,135 243 3,488 836 1,386 40 
 P11 8,566 6,834 12,490 13,618 2,054 4,164 42 
 P12 4,770 14,549 2,274 24,576 21,583 16,650 42 
         
Peptide+ IL-2 P13 2,141 121 1,735 853 1,992 283 
 P14 1,895 1,714 1,607 2,335 663 1,026 199 
 P15 1,842 9,524 1,791 10,043 1,519 4,941 89 
 P16 7,225 4,887 4,620 2,771 2,228 1,473 184 
 P17 433 300 533 14,496 6,309 45 
 P18 2,345 971 1,755 272 421 145 
 P19 616 271 1,051 251 1,690 1,656 54 
 P20 2,558 950 769 979 908 2,687 50 
 P21 1,870 239 895 306 838 260 94 
 P22 3,134 1,813 4,507 1,338 1,838 1,341 41 
 P23 2,427 1,057 2,426 869 1,374 465 85 
a

Frequency calculation: [URQ/(LRQ + URQ) × 10e6]epitope-specific − [URQ/(LRQ + URQ) × 10e6]background.

b

Staining with g209-, g209-2M-, or Flu-tHLA.

c

Time interval between the pre- and postvaccination sample.

d

Treatment group.

e

Calculated frequencies below or equal to zero were arbitrarily set to 1 to permit statistical analysis involving log10.

FIGURE 2.

Frequency differences of g209 tHLA+ CD8+ cells in different treatment groups. The log10 differences of frequency were calculated for each pre- and postvaccination sample pair and analyzed with the Wilcoxon signed rank test. The means of the log10 difference are depicted as thick bars; positive means indicate higher post- than prevaccination frequencies. The SEs of the mean (thin bars) are displayed symmetrically. As there was insufficient evidence for normal distribution in some sample groups, this figure is an approximation. The treatment groups were P alone (n = 7), P + IL-12 (n = 5), and P + IL-2 (n = 11). A, Analysis of PBMC on day 1. B and C, T cell cultures 10 days after in vitro stimulation with g209 or g209–2M peptide, respectively. The KW values indicate the significance of the difference among the three treatment groups.

FIGURE 2.

Frequency differences of g209 tHLA+ CD8+ cells in different treatment groups. The log10 differences of frequency were calculated for each pre- and postvaccination sample pair and analyzed with the Wilcoxon signed rank test. The means of the log10 difference are depicted as thick bars; positive means indicate higher post- than prevaccination frequencies. The SEs of the mean (thin bars) are displayed symmetrically. As there was insufficient evidence for normal distribution in some sample groups, this figure is an approximation. The treatment groups were P alone (n = 7), P + IL-12 (n = 5), and P + IL-2 (n = 11). A, Analysis of PBMC on day 1. B and C, T cell cultures 10 days after in vitro stimulation with g209 or g209–2M peptide, respectively. The KW values indicate the significance of the difference among the three treatment groups.

Close modal
FIGURE 4.

Frequency differences of Flu tHLA+ CD8+ cells in different treatment groups. The log10 differences of frequency were calculated for each pre- and postvaccination sample pair and analyzed with the Wilcoxon signed rank test. The means of the log10 difference are depicted as thick bars; positive means indicate higher post- than prevaccination frequencies. The SEs of the mean (thin bars) are displayed symmetrically. As there was insufficient evidence for normal distribution in some sample groups, this figure is an approximation. The treatment groups were P alone (n = 7), P + IL-12 (n = 5), and P + IL-2 (n = 11). A, Analysis of PBMC on day 1. B, T cell cultures 10 days after in vitro stimulation with Flu peptide. The KW values indicate the significance of the difference among the three treatment groups.

FIGURE 4.

Frequency differences of Flu tHLA+ CD8+ cells in different treatment groups. The log10 differences of frequency were calculated for each pre- and postvaccination sample pair and analyzed with the Wilcoxon signed rank test. The means of the log10 difference are depicted as thick bars; positive means indicate higher post- than prevaccination frequencies. The SEs of the mean (thin bars) are displayed symmetrically. As there was insufficient evidence for normal distribution in some sample groups, this figure is an approximation. The treatment groups were P alone (n = 7), P + IL-12 (n = 5), and P + IL-2 (n = 11). A, Analysis of PBMC on day 1. B, T cell cultures 10 days after in vitro stimulation with Flu peptide. The KW values indicate the significance of the difference among the three treatment groups.

Close modal

Because determination of vaccine-specific TCPF does not yield information about their ability to respond to epitope-specific stimulation, we performed an in vitro expansion of the same PBMC in all 23 patients as exemplified for patient 6 (Fig. 5). The selection of PBMC as APC emphasizes a bias toward expansion of memory T cells elicited by the vaccination. Furthermore, this method was selected because it is the one previously described for the assessment of response to vaccination. Thus, analysis of CTL expansion after in vitro stimulation using tHLA can be directly compared with results previously reported by our group obtained by evaluating cytokine release and cytotoxicity of vaccine-induced in vitro-expanded T cells (2, 3, 4). As shown in this patient, an enrichment of vaccine-specific T cells could be easily demonstrated after in vitro stimulation of post- but not prevaccination PBMC. Concomitant stimulation of T cells specific for an epitope irrelevant to the one used for in vitro stimulation was occasionally noted. In patient 6, we noted persistence and/or minor expansion of Flu-specific CTL in cultures stimulated with g209–2M. Conversely, g209 and g209–2M-specific T cells could be identified in postvaccination cultures stimulated with Flu. In no case could the concomitant T cell expansion be noted in unstimulated control cultures (data not shown).

FIGURE 5.

Comparison of TCPF determined on PBMC and after ex vivo stimulation. TCPF of pre- and postvaccination samples from P6 were determined in PBMC and compared with TCPF of 10-day cultures stimulated with g209, g209–2M, and Flu as specified by the open arrows. Titles of columns refer to the tHLA used for staining. PBMC were stained with g209 tHLA (first column, top two dot plots), g209–2M (first column, middle two dot plots), and Flu (first column, bottom two dot plots). T cell cultures stimulated with peptide were stained with g209 (second column), g209–2M (third column), and Flu (fourth column) tHLA. Numbers in the URQ indicate the percentage of tHLA staining CD8+ T cells calculated according to the formula: URQ/(URQ + LRQ) × 100.

FIGURE 5.

Comparison of TCPF determined on PBMC and after ex vivo stimulation. TCPF of pre- and postvaccination samples from P6 were determined in PBMC and compared with TCPF of 10-day cultures stimulated with g209, g209–2M, and Flu as specified by the open arrows. Titles of columns refer to the tHLA used for staining. PBMC were stained with g209 tHLA (first column, top two dot plots), g209–2M (first column, middle two dot plots), and Flu (first column, bottom two dot plots). T cell cultures stimulated with peptide were stained with g209 (second column), g209–2M (third column), and Flu (fourth column) tHLA. Numbers in the URQ indicate the percentage of tHLA staining CD8+ T cells calculated according to the formula: URQ/(URQ + LRQ) × 100.

Close modal

TCPF determined after 10 days of in vitro culture and after one stimulation with g209, g209–2M, or Flu peptides are shown in Tables II, III, and IV, respectively. After in vitro stimulation with g209 or g209–2M, all treatment groups demonstrated a peptide-specific expansion in the postvaccination samples. The TCPF increased dramatically in most patients treated with P alone or P + IL-12, and reached up to 29% of CD8+ cells (P12 in Table III). The increase was less pronounced in the P + IL-2 group. In fact, most patients in the P + IL-2 group did not show any increase. The amplified fc differences between pre- and postvaccination cultures underlined the trends observed in PBMC within each treatment group. However, fc differences in pre- and postvaccination PBMC of single patients were not reflected quantitatively by fc differences in the corresponding in vitro cultures.

Table III.

Calculated precursor frequenciesa of g209-2M-specific CD8+ cells on day 10 (peptide-specific/10e6 CD8+ cells)

d1g209 Stimulationbg209-2M Stimulation
PrePostPrePostPrePost
Peptide alonec P1 638 9,664 427 7,203 986 11,271 
 P2 2,218 4,916 749 798 1,582 4,576 
 P3 1,558 4,301 674 129,367 854 113,703 
 P4 2,587 23,952 283 249,081 280 296,596 
 P5 1,416 3,440 537 15,161 2,780 60,936 
 P6 5,473 12,723 3,842 248,973 1,000 287,991 
 P7 408 1,564 289 3,140 318 13,412 
        
Peptide+ IL-12 P8 500 5,609 1d 137,740 1,202 226,460 
 P9 401 1,714 422 4,935 1,085 43,216 
 P10 243 3,488 540 3,291 710 19,966 
 P11 12,490 13,618 1,318 58,345 696 72,815 
 P12 2,274 24,576 287,954 2,058 291,347 
        
Peptide+ IL-2 P13 1,735 853 22,517 25,930 26,037 19,794 
 P14 1,607 2,335 3,849 4,860 7,221 2,924 
 P15 1,791 10,043 873 5,313 563 11,763 
 P16 4,620 2,771 872 2,877 1,863 3,066 
 P17 300 533 365 1,227 270 1,502 
 P18 1,755 272 18,272 23,677 7,404 15,465 
 P19 1,051 251 753 3,466 355 3,379 
 P20 769 979 2,855 3,466 1,385 3,379 
 P21 895 306 556 16,699 686 41,414 
 P22 4,507 1,338 754 935 349 715 
 P23 2,426 869 542 3,738 260 10,406 
d1g209 Stimulationbg209-2M Stimulation
PrePostPrePostPrePost
Peptide alonec P1 638 9,664 427 7,203 986 11,271 
 P2 2,218 4,916 749 798 1,582 4,576 
 P3 1,558 4,301 674 129,367 854 113,703 
 P4 2,587 23,952 283 249,081 280 296,596 
 P5 1,416 3,440 537 15,161 2,780 60,936 
 P6 5,473 12,723 3,842 248,973 1,000 287,991 
 P7 408 1,564 289 3,140 318 13,412 
        
Peptide+ IL-12 P8 500 5,609 1d 137,740 1,202 226,460 
 P9 401 1,714 422 4,935 1,085 43,216 
 P10 243 3,488 540 3,291 710 19,966 
 P11 12,490 13,618 1,318 58,345 696 72,815 
 P12 2,274 24,576 287,954 2,058 291,347 
        
Peptide+ IL-2 P13 1,735 853 22,517 25,930 26,037 19,794 
 P14 1,607 2,335 3,849 4,860 7,221 2,924 
 P15 1,791 10,043 873 5,313 563 11,763 
 P16 4,620 2,771 872 2,877 1,863 3,066 
 P17 300 533 365 1,227 270 1,502 
 P18 1,755 272 18,272 23,677 7,404 15,465 
 P19 1,051 251 753 3,466 355 3,379 
 P20 769 979 2,855 3,466 1,385 3,379 
 P21 895 306 556 16,699 686 41,414 
 P22 4,507 1,338 754 935 349 715 
 P23 2,426 869 542 3,738 260 10,406 
a

Frequency calculation: [URQ/(LRQ + URQ) × 10e6]epitope-specific − [URQ/(LRQ + URQ) × 10e6]background.

b

Ten-day in vitro cultures stimulated with g209 or g209-2M.

c

Treatment group.

d

Calculated frequencies below or equal to zero were arbitrarily set to 1 to permit statistical analysis involving log10. Control cultures stimulated with Flu or without any peptide did not demonstrate an increase in g209/g209-2M-specific TCPF (data not shown).

Statistically significant differences in specific T cell expansion between pre- and postvaccination samples could be detected in the patient group receiving P alone (Fig. 2, B and C; Fig. 3, B and C, and Fig. 4 B). The P + IL-12 group demonstrated the same trend but did not reach statistical significance. Measurement of vaccine-specific T cell expansion in patients receiving peptide plus IL-2 also revealed a modest but significant increase in post- compared with prevaccination samples. Thus, it appears that the inability to detect an enhancement of vaccine-specific T cell in these patients was in part due to a limitation in the sensitivity of tHLA staining rather than an absolute lack of effect of the vaccine. This analysis, therefore, demonstrates that the enhancement of TCPF detected in PBMC in response to vaccination is associated with an enhanced susceptibility to in vitro epitope-specific stimulation.

FIGURE 3.

Frequency differences of g209–2M tHLA+ CD8+ cells in different treatment groups. The log10 differences of frequency were calculated for each pre- and postvaccination sample pair and analyzed with the Wilcoxon signed rank test. The means of the log10 difference are depicted as thick bars; positive means indicate higher post- than prevaccination frequencies. The SEs of the mean (thin bars) are displayed symmetrically. As there was insufficient evidence for normal distribution in some sample groups, this figure is an approximation. The treatment groups were P alone (n = 7), P + IL-12 (n = 5), and P + IL-2 (n = 11). A, Analysis of PBMC on day 1. B and C, T cell cultures 10 days after in vitro stimulation with g209 or g209–2M peptide respectively. The KW values indicate the significance of the difference among the three treatment groups.

FIGURE 3.

Frequency differences of g209–2M tHLA+ CD8+ cells in different treatment groups. The log10 differences of frequency were calculated for each pre- and postvaccination sample pair and analyzed with the Wilcoxon signed rank test. The means of the log10 difference are depicted as thick bars; positive means indicate higher post- than prevaccination frequencies. The SEs of the mean (thin bars) are displayed symmetrically. As there was insufficient evidence for normal distribution in some sample groups, this figure is an approximation. The treatment groups were P alone (n = 7), P + IL-12 (n = 5), and P + IL-2 (n = 11). A, Analysis of PBMC on day 1. B and C, T cell cultures 10 days after in vitro stimulation with g209 or g209–2M peptide respectively. The KW values indicate the significance of the difference among the three treatment groups.

Close modal

In one patient (P4), TCPF was monitored throughout treatment with multiple vaccinations (before and after 1, 2, 4, 6, 8, and 10 vaccinations) (Fig. 6). One vaccination was not sufficient to elicit a detectable increase in TCPC. However, a strong enhancement was noted 3 wk after the second vaccination (Fig. 1) at which time point there was no evidence of tumor regression. Starting from the third vaccination, IL-2 was added. Surprisingly, the TCPF decreased and tumors began to shrink. Thus, the detection of vaccine-specific T cells in PBMC after two vaccinations did not correlate with clinical outcome. Rapid expansion in response to vaccine-specific stimulation in vitro makes it unlikely that functional unresponsiveness was responsible for tumor progression. To more directly assess the functional state of g209/g209–2M-specific T cell, pre- and postvaccination PBMC were compared for intracellular IFN-γ expression in response to stimulation with T2 cells exogenously pulsed with g209 or g209–2M (Fig. 7). This analysis demonstrated a specific enhancement of g209/g209–2M reactive T cells only in postvaccination PBMC, which correlated with specific down-regulation of TCR as judged by tetramer staining. Interestingly the number of IFN-γ-expressing T cells in the postvaccination PBMC sample was noted to be roughly half the number of tetramer positive T cells. It is possible that tetramer staining of postvaccination PBMC picks up a fraction of vaccine-induced T cells that do not produce IFN-γ in response to Ag stimulation in the in vitro conditions exercised used for this study.

FIGURE 6.

Evaluation of vaccine-specific T cell fc in a patient throughout an entire vaccination treatment. The frequency of vaccine-specific T cell is shown at different time points. Number of vaccination refers to the vaccine that had been administered before the analysis of a given specimen. The arrow shows the period in which IL-2 was added to the patient’s treatment. The PBMC samples were stained with g209–2M and g209 tHLA as well as Flu tHLA. Flu-specific TCPF did not change significantly throughout the vaccination treatment (data not shown).

FIGURE 6.

Evaluation of vaccine-specific T cell fc in a patient throughout an entire vaccination treatment. The frequency of vaccine-specific T cell is shown at different time points. Number of vaccination refers to the vaccine that had been administered before the analysis of a given specimen. The arrow shows the period in which IL-2 was added to the patient’s treatment. The PBMC samples were stained with g209–2M and g209 tHLA as well as Flu tHLA. Flu-specific TCPF did not change significantly throughout the vaccination treatment (data not shown).

Close modal
FIGURE 7.

FACS analysis for intracellular IFN-γ expression in pre- and postvaccination PBMC from patient 4. The PBMC were obtained after two vaccinations before that patient had received concomitant systemic IL-2 therapy and were stimulated with unpulsed T2 cells (T2(−)), or pulsed with g209 (T2(g209)) or g2092M (T2(g209–2M)) peptides (1 μM). CD3+/CD8+ cells were gated for analysis and numbers indicate the percentage of cells in the quadrants over the total gated cells. g209 tHLA staining decreased upon stimulation due to down-regulation of TCR, as previously noted analyzing epitope-specific clonal populations (data not shown).

FIGURE 7.

FACS analysis for intracellular IFN-γ expression in pre- and postvaccination PBMC from patient 4. The PBMC were obtained after two vaccinations before that patient had received concomitant systemic IL-2 therapy and were stimulated with unpulsed T2 cells (T2(−)), or pulsed with g209 (T2(g209)) or g2092M (T2(g209–2M)) peptides (1 μM). CD3+/CD8+ cells were gated for analysis and numbers indicate the percentage of cells in the quadrants over the total gated cells. g209 tHLA staining decreased upon stimulation due to down-regulation of TCR, as previously noted analyzing epitope-specific clonal populations (data not shown).

Close modal

After eight vaccinations, the patient developed a new s.c. metastasis. Analysis of T cells obtained from a fine needle aspirate of that lesion failed to demonstrate evidence of localization of vaccine-specific CD8+ T cells at the tumor site. Immunocytochemical analysis of the tumor at this time point revealed loss of gp100, which might explain the clinical outcome and, at the same time, the failure to identify vaccine-specific T cells.

The low TCPF observed in the P + IL-2 group, which has been associated with clinical responses, may be explained by a migration of tumor-specific T cells from the systemic circulation to the tumor site. To address whether this phenomenon really occurs, we analyzed PBMC and tumor preparations, which had been obtained at the same time point after vaccination. Seven such pairs were available for analysis (Table V). In two of seven cases (L.R. and D.W.), a suggestive evidence of localization was seen. Interestingly, these lesions demonstrated high expression of the target Ag gp100 by immunocytochemistry. It should be emphasized, however, that this analysis could be strongly biased by the selection of lesions that had persisted after immunization.

Table V.

Calculated precursor frequenciesa of g209-specific CD8+ cells in PBMC/tumor pairs (g209-specific/10e6 CD8+ cells)

Patientgp100HLA-A2PBMCTumor
D.G. NAb NA 279 63 
F.K. Negative Positive 2283 3492 
L.R. >75%, 2+ Positive 528 3134 
J.R. NA NA 249 0c 
D.W. >75%, 3+ Positive 596 2448 
A.C. >75%, 2+ Positive 1545 659 
S.B. <25%, 2+ Positive 563 
Patientgp100HLA-A2PBMCTumor
D.G. NAb NA 279 63 
F.K. Negative Positive 2283 3492 
L.R. >75%, 2+ Positive 528 3134 
J.R. NA NA 249 0c 
D.W. >75%, 3+ Positive 596 2448 
A.C. >75%, 2+ Positive 1545 659 
S.B. <25%, 2+ Positive 563 
a

Frequency calculation: [URQ/(LRQ + URQ) × 10e6]epitope-specific − [URQ/(LRQ + URQ) × 10e6]background.

b

NA, not available.

c

Calculated frequencies below or equal to zero were arbitrarily set to 0.

Systemic and local T cell-based immune responses against melanoma have been repeatedly identified in humans (1, 13, 14, 15, 16, 17). Rarely, however, are detectable immune responses sufficient to arrest tumor growth as apparent by the grim prognosis of advanced melanoma and the frequent identification of immune reactivity in patients whose disease is rapidly leading to their demise. Yet the ease with which MA-specific TIL can be generated from tumors (13, 14) illustrates the awareness of cancer cells by the immune system. Thus, a paradoxical coexistence of immune competent T cells and their respective targets appears to occur in vivo as judged from reagents characterized ex vivo.

The identification of MA epitopes recognized by T cells has led to the utilization of minimal peptide sequences for the in vivo induction or amplification of systemic, tumor-specific T cell responses (1, 2). Results from pilot studies testify for the high specificity of the induced T cell responses against HLA-matched tumor cells (2, 3, 4). These studies gave the impression that vaccines induce powerful immunizations comparable to those demonstrable against common pathogens such as the influenza virus to which individuals are repeatedly exposed throughout their lifetime. In most cases, this vaccine-induced T cell reactivity still does not lead to tumor regression.

Most studies thus far have measured T cell response to epitope-specific vaccination by comparative assessment of pre- and postvaccination PBMC for tumor-specific T cell expansion in vitro in response to stimulation with relevant and irrelevant epitopes, as originally described by Vitiello et al. (18) in a viral system. These assays are excellent for qualitative assessment of T cell responses at two time points in an individual’s life or for nonparametric comparison of treatment outcomes in different patient groups. However, they cannot provide quantitative insight about the strength of the observed response because of the arbitrary nature of the stimulus applied ex vivo and the addition of secondary proliferative stimuli, which most commonly consists of IL-2. Thus, as for TIL, it is likely that the immune responses judged after ex vivo expansion of postvaccination PBMC overestimate quantitatively the strength of the immune reaction within the organism.

Therefore, other methods of analysis have been fashioned to evaluate T cell responses directly without the need for ex vivo amplification. Among these, the ELISPOT assay has enjoyed notable popularity because of its simplicity, relative accuracy and sensitivity (19). Analysis of postvaccination PBMC from patients treated with g209–2M peptide in IFA could identify T cells specific for the altered epitope in four of six patients. In the four patients, the estimated frequencies ranged between 1/1000 and 1/2000 epitope-specific T cells (6). In no patient was it possible to identify T cells recognizing the natural epitope (g209) by the ELISPOT assay, whereas limiting dilution assays estimated precursor frequencies for g209-reactive T cells to range between 1/3000 and 1/6000 (2). Because of the dependency upon cytokine secretion/proliferation, these assays may underestimate the actual frequency of CTL precursors by not identifying T cells with a threshold for cytokine expression/proliferation above the stimulus applied in the assays (10). Moreover, naive T cells, less responsive to epitope-specific stimulation, might be missed by these functional assays (20). Recently, the use of HLA/epitope tetramers (7) has offered a tool to directly measure the frequency of CTL precursors presumably independently of their functional state. Measurements by this assay demonstrated CTL precursor frequencies considerably higher than those suggested by ELISPOT or limiting dilution assays (10).

T cell responses to epitope-specific vaccination have not been measured with this direct assay, yet. Several studies have measured the response of T cells to acute or chronic viral infections or during ongoing autoimmune episodes (8, 9, 10, 21, 22, 23). So far studies on tumor reactivity have been presented by few groups of investigators and have been primarily limited to the analysis of MART-1 and tyrosinase. In melanoma patients with vitiligo, MART-1-specific CTL have been identified at a frequency up to 0.67% of CD8+ T cells (24). Furthermore, MART-1-specific T cells could be identified in melanoma infiltrated lymph nodes with a frequency ranging from 0.22 to 1.8% of CD8+ T cells and correlated with MA expression (25). Characterization of circulating T cells demonstrated identifiable MA-specific T cells in approximately half of patients affected with metastatic melanoma (26). Functional characterization of MA-specific T cells from one patient lead to the generalization that some of the T cells identified with tHLA are unresponsive to Ag stimulation and, thus, unable to control tumor growth. This study is the first quantitative evaluation of the response to an HLA class I-restricted epitope vaccination. Epitope-specific vaccination yielded significant differences between the pre- and posttreatment CTL precursor frequencies. Vaccine-specific T cell frequency increased up to 2.5% of CD8+ cells after vaccination, and the frequency of T cell recognizing the natural gp100 epitope (g209) was enhanced up to 1.4%. In a significant proportion of patients, frequency of g209 recognizing T cell after vaccination ranged between 0.2 to 0.9%. Thus, a significant conceptual finding of this study is the limited extent of vaccine-specific response. We were surprised at the relatively low numbers of CTL precursors after vaccination even in patients’ samples that boasted an exceptional epitope-specific expansion in vitro. An inverse correlation has been reported between HIV-specific CTL frequency and viral RNA load in HIV infected individuals (8). Furthermore, TCPF as high as 2% of CD8+ T cells have been reported in HIV-infected patients, who remained asymptomatic (22). Thus, it is possible that the immune response elicited by the vaccination regimen used in this study did not reach the quantitative capacity necessary for tumor regression.

Clonal deletion, exhaustion, or senescence (27, 28, 29, 30, 31, 32, 33, 34) have been implicated in the induction of systemic, epitope-specific immune tolerance. However, because g209-specific T cell could be identified after vaccination in this study, deletion of tumor-reactive T cells may not be as significant in humans as suggested by preclinical models (27, 30, 33, 34, 35). Inadequate immune responses in patients with cancer and other chronic illnesses have been attributed to decreased TCR signaling capacity (36, 37) or circulating immune-suppressive cytokines (37). Finally, analysis of MA-specific T cells from one patient has lead to the generalization that tumor-specific T cells may be anergic in vivo (26). However, in this study, T cells elicited by the vaccine could be readily stimulated with the cognate epitope to rapidly proliferate in culture. Furthermore, analysis of postvaccination PBMC identified a significant percentage of vaccine-specific T cells capable to secrete IFN-γ in response to vaccine-specific stimulation. Taken together, these data suggest that the extent rather than the quality of the response might be the more significant limitation of the vaccination protocol analyzed in this study.

Although differences in TCPF were detectable between pre- and postvaccination PBMC selected from patients that had been treated with peptide alone, no significant differences could be detected in patients who had received peptide plus systemic IL-2 therapy and had shown no enhanced reactivity by in vitro sensitization assays (2). The association of undetectable vaccine-specific T cells with the enhanced frequency of clinical responses after systemic administration of IL-2 remains mysterious. One possibility is that the responsiveness of T cell to in vitro restimulation might be reduced in patients who received IL-2. This study demonstrates instead that the number rather than susceptibility to in vitro expansion is decreased in these patients. It has been suggested that tumors will induce tolerance by presenting epitope-specific stimulation (signal one) without costimulation (signal two) to wandering MA-recognizing T cells (38). By increasing vascular permeability, IL-2 might facilitate encounters between T cells and cancer cells that lead to reduced tumor burden and T cell number at the same time. Study of seven simultaneously obtained PBMC and tumor samples demonstrated a slightly increased frequency of vaccine-induced T cells at tumor site in only two of the pairs studied in correlation with expression of gp100. It is possible that insufficient localization and/or poor survival of vaccine-induced T cells at tumor site may be the reason why the lesions analyzed in this study did not regress in response to therapy.

Table II.

Calculated precursor frequenciesa of g209-specific CD8+ cells on day 10 (peptide-specific/10e6 CD8+ cells)

d1g209 Stimulationbg209-2M Stimulation
PrePostPrePostPrePost
Peptide alonec P1 356 2,109 538 18,291 429 20,655 
 P2 667 2,863 2,895 2,323 1,211 2,595 
 P3 2,083 3,451 460 133,698 585 98,962 
 P4 1,854 14,806 189 196,025 379 197,705 
 P5 104 4,402 540 12,206 1,569 16,637 
 P6 4,779 9,137 4,036 240,755 741 231,598 
 P7 454 1,988 175 971 370 3,779 
        
Peptide+ IL-12 P8 508 4,177 1d 107,871 613 123,606 
 P9 497 2,199 659 4,018 1,015 42,937 
 P10 282 3,135 268 1,078 586 6,242 
 P11 8,566 6,834 1,321 47,490 279 73,518 
 P12 4,770 14,549 184,158 1,397 146,721 
        
Peptide+ IL-2 P13 2,141 121 16,140 25,141 16,304 13,802 
 P14 1,895 1,714 3,519 2,587 4,370 3,538 
 P15 1,842 9,524 1,073 4,203 233 9,625 
 P16 7,225 4,887 2,283 3,417 1,654 1,282 
 P17 433 628 987 596 1,123 
 P18 2,345 971 16,380 19,702 6,425 11,965 
 P19 616 271 2,949 129 725 
 P20 2,558 950 3,663 2,039 1,388 725 
 P21 1,870 239 564 7,454 182 14,645 
 P22 3,134 1,813 1,142 1,327 172 508 
 P23 2,427 1,057 124 2,129 3,023 
d1g209 Stimulationbg209-2M Stimulation
PrePostPrePostPrePost
Peptide alonec P1 356 2,109 538 18,291 429 20,655 
 P2 667 2,863 2,895 2,323 1,211 2,595 
 P3 2,083 3,451 460 133,698 585 98,962 
 P4 1,854 14,806 189 196,025 379 197,705 
 P5 104 4,402 540 12,206 1,569 16,637 
 P6 4,779 9,137 4,036 240,755 741 231,598 
 P7 454 1,988 175 971 370 3,779 
        
Peptide+ IL-12 P8 508 4,177 1d 107,871 613 123,606 
 P9 497 2,199 659 4,018 1,015 42,937 
 P10 282 3,135 268 1,078 586 6,242 
 P11 8,566 6,834 1,321 47,490 279 73,518 
 P12 4,770 14,549 184,158 1,397 146,721 
        
Peptide+ IL-2 P13 2,141 121 16,140 25,141 16,304 13,802 
 P14 1,895 1,714 3,519 2,587 4,370 3,538 
 P15 1,842 9,524 1,073 4,203 233 9,625 
 P16 7,225 4,887 2,283 3,417 1,654 1,282 
 P17 433 628 987 596 1,123 
 P18 2,345 971 16,380 19,702 6,425 11,965 
 P19 616 271 2,949 129 725 
 P20 2,558 950 3,663 2,039 1,388 725 
 P21 1,870 239 564 7,454 182 14,645 
 P22 3,134 1,813 1,142 1,327 172 508 
 P23 2,427 1,057 124 2,129 3,023 
a

Frequency calculation: [URQ/(LRQ + URQ) × 10e6]epitope-specific − [URQ/(LRQ + URQ) × 10e6]background.

b

Ten-day in vitro cultures stimulated with g209 or g209-2M.

c

Treatment group.

d

Calculated frequencies below or equal to zero were arbitrarily set to 1 to permit statistical analysis involving log10. Control cultures stimulated with Flu or without any peptide did not demonstrate an increase in g209/g209-2M-specific TCPF (data not shown).

Table IV.

Calculated precursor frequenciesa of Flu-specific CD8+ cells on day 10 (peptide-specific/10e6 CD8+ cells)

d1Flu Stimulationb
PrePostPrePost
Peptide alonec P1 357 2,254 7,280 6,538 
 P2 2,177 3,064 19,347 33,450 
 P3 1,115 1,274 497 1,334 
 P4 2,870 1d 43,229 72,034 
 P5 4,732 5,703 17,348 18,676 
 P6 6,820 7,901 173,757 131,309 
 P7 415 2,389 30,492 32,211 
      
Peptide+ IL-12 P8 2,002 1,237 46,275 27,266 
 P9 480 667 12,150 12,462 
 P10 836 1,386 436 13,785 
 P11 2,054 4,164 15,343 30,713 
 P12 21,583 16,650 228,989 121,649 
      
Peptide+ IL-2 P13 1,992 8,189 8,288 
 P14 663 1,026 63,546 21,965 
 P15 1,519 4,941 43,607 78,601 
 P16 2,228 1,473 34,709 2,864 
 P17 14,496 6,309 75,455 99,607 
 P18 421 1,612 2,285 
 P19 1,690 1,656 38,732 53,972 
 P20 908 2,687 20,011 53,972 
 P21 838 260 65,978 115,133 
 P22 1,838 1,341 7,071 7,373 
 P23 1,374 465 261 4,267 
d1Flu Stimulationb
PrePostPrePost
Peptide alonec P1 357 2,254 7,280 6,538 
 P2 2,177 3,064 19,347 33,450 
 P3 1,115 1,274 497 1,334 
 P4 2,870 1d 43,229 72,034 
 P5 4,732 5,703 17,348 18,676 
 P6 6,820 7,901 173,757 131,309 
 P7 415 2,389 30,492 32,211 
      
Peptide+ IL-12 P8 2,002 1,237 46,275 27,266 
 P9 480 667 12,150 12,462 
 P10 836 1,386 436 13,785 
 P11 2,054 4,164 15,343 30,713 
 P12 21,583 16,650 228,989 121,649 
      
Peptide+ IL-2 P13 1,992 8,189 8,288 
 P14 663 1,026 63,546 21,965 
 P15 1,519 4,941 43,607 78,601 
 P16 2,228 1,473 34,709 2,864 
 P17 14,496 6,309 75,455 99,607 
 P18 421 1,612 2,285 
 P19 1,690 1,656 38,732 53,972 
 P20 908 2,687 20,011 53,972 
 P21 838 260 65,978 115,133 
 P22 1,838 1,341 7,071 7,373 
 P23 1,374 465 261 4,267 
a

Frequency calculation: [URQ/(LRQ + URQ) × 10e6]epitope-specific − [URQ/(LRQ + URQ) × 10e6]background.

b

Ten-day in vitro cultures stimulated with Flu.

c

Treatment group.

d

Calculated frequencies below or equal to zero were arbitrarily set to 1 to permit statistical analysis involving log10. No increase in g209/g209-2M-specific TCPF (data not shown).

2

Abbreviations used in this paper: MA, melanoma Ag; tHLA, tetramer HLA; g209, gp100:209–217; g209–2M, gp100:209–217(210M); ELISPOT, enzyme-linked immunospot; MART-1, MART-1:26–35 (27L); Flu, FluM1:58–66; TCPF, precursor frequency; TIL, tumor infiltrating lymphocyte; fc, calculated frequency; KW, Kruskal-Wallis; P, peptide.

1
Rosenberg, S. A..
1997
. Cancer vaccines based on the identification of genes encoding cancer regression antigens.
Immunol. Today
18
:
175
2
Rosenberg, S. A., J. C. Yang, D. Schwartzentruber, P. Hwu, F. M. Marincola, S. L. Topalian, N. P. Restifo, E. Dufour, L. Schwartzberg, P. Spiess, et al
1998
. Immunologic and therapeutic evaluation of a synthetic tumor associated peptide vaccine for the treatment of patients with metastatic melanoma.
Nat. Med.
4
:
321
3
Cormier, J. N., M. L. Salgaller, T. Prevette, K. C. Barracchini, L. Rivoltini, N. P. Restifo, S. A. Rosenberg, F. M. Marincola.
1997
. Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A.
Cancer J. Sci. Am.
3
:
37
4
Salgaller, M. L., F. M. Marincola, J. N. Cormier, S. A. Rosenberg.
1996
. Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.
Cancer Res.
56
:
4749
5
Parkhurst, M. R., M. L. Salgaller, S. Southwood, P. F. Robbins, A. Sette, S. A. Rosenberg, Y. Kawakami.
1996
. Improved induction of melanoma reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201 binding residues.
J. Immunol.
157
:
2539
6
Pass, H. A., S. L. Schwarz, J. R. Wunderlich, S. A. Rosenberg.
1998
. Immunization of patients with melanoma peptide vaccines: immunologic assessment using the ELISPOT assay.
Cancer J. Sci. Am.
4
:
316
7
Altman, J. D., P. H. Moss, P. R. Goulder, D. H. Barouch, M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, M. M. Davis.
1996
. Phenotypic analysis of antigen-specific T lymphocytes.
Science
274
:
94
8
Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard, J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al
1998
. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.
Science
279
:
2103
9
Callan, M. F., L. Tan, N. Annels, G. S. Ogg, J. D. Wilson, C. A. O’Callaghan, N. Steven, A. J. McMichael, A. B. Rickinson.
1998
. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo.
J. Exp. Med.
187
:
1395
10
Tan, L. C., N. Gudgeon, N. E. Annels, P. Hansasuta, C. A. O’Callaghan, S. Rowland-Jones, A. J. McMichael, A. B. Rickinson, M. C. F. Callan.
1999
. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers.
J. Immunol.
162
:
1827
11
Bunce, M., C. M. O’Neill, M. C. Barnardo, P. Krausa, M. J. Browning, P. J. Morris, K. I. Welsh.
1995
. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP).
Tissue Antigens
46
:
355
12
Krausa, P., M. Brywka, D. Savage, K. M. Hui, M. Bunce, J. L. Ngai, D. L. Teo, Y. W. Ong, D. Barouch, C. E. Allsop, et al
1995
. Genetic polymorphism within HLA-A*02: significant allelic variation revealed in different populations.
Tissue Antigens
45
:
223
13
Topalian, S. L., D. Solomon, S. A. Rosenberg.
1989
. Tumor-specific cytolysis by lymphocytes infiltrating human melanomas.
J. Immunol.
142
:
3714
14
Wolfel, T., E. Klehmann, C. Muller, K. H. Schutt, K. H. Meyer zum Buschenfelde, A. Knuth.
1989
. Lysis of human melanoma cells by autologous cytolytic T cell clones: identification of human histocompatibility leukocyte antigen A2 as a restriction element for three different antigens.
J. Exp. Med.
170
:
797
15
van der Bruggen, P., C. Traversari, P. Chomez, C. Lurquin, E. De Plaen, B. Van den Eynde, A. Knuth, T. Boon.
1991
. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
Science
254
:
1643
16
Boon, T., P. G. Coulie, B. Van den Eynde.
1997
. Tumor antigens recognized by T cells.
Immunol. Today
18
:
267
17
Old, L. J., Y. T. Chen.
1998
. New paths in human cancer serology.
J. Exp. Med.
187
:
1163
18
Vitiello, A., G. Ishioka, H. M. Grey, R. Rose, P. Farness, R. LaFond, L. Yuan, F. V. Chisari, J. Furze, R. Bartholomeuz, et al
1995
. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans.
J. Clin. Invest.
95
:
341
19
Scheibenbogen, C., K. H. Lee, S. Stevanovic, M. Witzens, M. Willhauck, V. Waldmann, H. Naeher, H. G. Rammensee, U. Keilholz.
1997
. Analysis of the T cell response to tumor and viral peptide antigens by an IFN-γ-ELISPOT assay.
Int. J Cancer
71
:
932
20
Dutton, R. W., L. M. Bradley, S. L. Swain.
1998
. T cell memory.
Annu. Rev. Immunol.
16
:
201
21
Kuroda, M. J., J. E. Schmitz, D. H. Barouch, A. Craiu, T. M. Allen, A. Sette, D. I. Watkins, M. A. Forman, N. L. Letvin.
1998
. Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.
J. Exp. Med.
187
:
1373
22
Gray, C. M., J. Lawrence, J. M. Schapiro, J. D. Altman, M. A. Winters, M. Crompton, M. Loi, S. K. Kundu, M. M. Davis, T. C. Merigan.
1999
. Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART).
J. Immunol.
162
:
1780
23
Bieganowska, K., P. Hollsberg, G. J. Buckle, D. G. Lim, T. F. Greten, J. Schneck, J. D. Altman, S. Jacobson, S. L. Ledis, B. Hanchard, et al
1999
. Direct analysis of viral-specific CD8+ T cells with soluble HLA-A2/Tax 11–19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy.
J. Immunol.
162
:
1765
24
Ogg, G. S., D. P. Rod, P. Romero, J. L. Chen, V. Cerundolo.
1998
. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.
J. Exp. Med.
188
:
1203
25
Romero, P., P. R. Dunbar, D. Valmori, M. Pittet, G. S. Ogg, D. Rimoldi, J. L. Chen, D. Lienard, J. C. Cerottini, V. Cerundolo.
1998
. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes.
J. Exp. Med.
188
:
1641
26
Lee, P. P., C. Yee, P. A. Savage, L. Fong, D. Brockstedt, J. S. Weber, D. Johnson, S. Swetter, J. Thompson, P. D. Greenberg, et al
1999
. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.
Nat. Med.
5
:
677
27
Lauritzsen, G. F., P. O. Hofgaard, K. Schenck, B. Bogen.
1998
. Clonal deletion of thymocytes as a tumor escape mechanism.
Int. J. Cancer
78
:
216
28
Toes, R. E., R. J. Blom, R. Offringa, W. M. Kast, C. J. Melief.
1996
. Enhanced tumor outgrowth after peptide vaccination: functional deletion of tumor-specific CTL induced by peptide vaccination can lead to the inability to reject tumors.
J. Immunol.
156
:
3911
29
Van Parijs, L., A. K. Abbas.
1998
. Homeostasis and self-tolerance in the immune system: turning lymphocytes off.
Science
280
:
243
30
Moskophidis, D., F. Lechner, H. P. Pircher, R. M. Zinkernagel.
1993
. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells.
Nature
362
:
758
31
Alexander-Miller, M. A., G. R. Leggatt, A. Sarin, J. A. Berzofsky.
1996
. Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.
J. Exp. Med.
184
:
485
32
Toes, R. E., S. P. Schoenberger, E. I. van der Voort, W. M. Kast, R. C. Hoeben, C. J. Melief, R. Offringa.
1997
. Activation or frustration of anti-tumor responses by T-cell-based immune modulation.
Semin. Immunol.
9
:
323
33
Effros, R. B., G. Pawelec.
1997
. Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion?.
Immunol. Today
18
:
450
34
Effros, R. B., R. Allsopp, C. P. Chiu, M. A. Hausner, K. Hirji, L. Wang, C. B. Harley, B. Villeponteau, M. D. West, J. V. Giorgi.
1996
. Shortened telomeres in the expanded CD28CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis.
AIDS
10
:
F17
35
Toes, R. E., R. Offringa, R. J. Blom, C. J. Melief, W. M. Kast.
1996
. Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.
Proc. Natl. Acad. Sci. USA
93
:
7855
36
Zea, A. H., B. D. Curti, D. L. Longo, W. G. Alvord, S. L. Strobl, H. Mizoguchi, S. P. Creekmore, J. J. O’Shea, G. C. Powers, W. J. Urba, A. C. Ochoa.
1995
. Alterations in T cell receptor and signal transduction molecules in melanoma patients.
Clin. Cancer Res.
1
:
1327
37
Wojtowicz-Praga, S..
1997
. Reversal of tumor-induced immunosuppression: a new approach to cancer therapy.
J. Immunother.
20
:
165
38
Matzinger, P..
1998
. An innate sense of danger.
Semin. Immunol.
10
:
399