Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-γ expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-γ expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

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The percentage of lesions not expressing the gp100 antigen is greater than the percentage not expressing MART-1 (26% vs 14%). The frequency of lesions with high expression (> 75%) of gp100 significantly decreased with therapy (47% vs 34%) and conversely negative lesions increased (18% vs 29%). Treatment of lesions with peptide alone (no IL-2) revealed a significant decrease in gp100 expression (47% vs 32%), enhanced with the addition of IL-2 to therapy (47% vs 35%). The expression of MART-1 remained essentially unchanged unless IL-2 was added (54% vs 54%, MART-1 peptide alone, 54% vs 43%, MART-1 peptide + IL-2). Of 94 patients (181 lesions) assessed for gp100 expression before treatment, 10 patients responded to therapy. Pretreatment lesions in responding patients expressed some level of gp100 in all cases compared with 27% of nonresponding lesions, which were negative for gp100 expression. CONCLUSIONS. This study indirectly demonstrates that vaccination with an MA-derived peptide can result in immune selection in vivo. Furthermore, it provides strong immunologic evidence for the specificity of MA vaccines and to the relevance of MA expression in predicting the response to vaccination.

We have established a sensitive ELISPOT assay measuring interferon gamma (IFN gamma) release on a single-cell basis to detect influenza peptide-specific CD8+ T cells in uncultured peripheral blood mononuclear cells (PBMC). Using this method, we studied the T cell response to HLA-A1 and HLA-A2.1 binding peptide epitopes derived from the MAGE-1 and MAGE-3 proteins, from the melanoma-associated antigens tyrosinase, Melan-A/MART-1 and gp100, and from influenza proteins in stage IV melanoma patients and healthy controls. In 18 of 24 HLA-A2-positive donors (75%), but only in 9 of 25 HLA-A2-positive melanoma patients (36%) T cells reactive with the influenza matrix peptide were demonstrated (p = 0.007). T cells responding to one or several of the melanoma-associated peptides were detected in 5 of 25 HLA-A2-positive patients with metastatic melanoma. Four of these 5 patients had been treated with interleukin-2- and IFN alpha-containing therapy. Two of the 24 healthy donors had T cells reactive with the MART-1 27-35 peptide. No reactivity with the HLA-A1-binding peptides from MAGE-1 or MAGE-3 was detected in any of the HLA-A1-positive healthy controls or melanoma patients. These results show that the IFN gamma-ELISPOT assay is suitable to determine quantitatively T cells reactive with melanoma-associated and influenza peptide epitopes in uncultured PBMC. The failure to detect T cells responding to influenza in many melanoma patients with progressive disease may indicate an impairment of their T cell function.