Dendritic cell immunizations alone or combined with low doses of interleukin-2 induce specific immune responses in melanoma patients

Abstract

Dendritic cell (DC)-based therapy has proved to be effective in patients with a variety of malignancies. However, an optimal immunization protocol using DCs and the best means for delivering antigens has not yet been described. In this study, 20 patients with malignant melanoma in stages III or IV were vaccinated with autologous DCs pulsed with a melanoma cell lysate, alone (n = 13) or in combination with low doses of subcutaneous (s.c.) interleukin (IL)-2 injections (n = 7), to assess toxicity, immunological and clinical responses. Monocyte-derived DCs were morphological, phenotypic and functionally characterized in vitro. Peripheral blood mononuclear cells (PBMC), harvested from patients either prior to and after the treatment, were analysed using enzyme-linked immunosorbent spot (ELISPOT). After vaccination, 50% of the patients tested (seven of 13) from the first group and (three of seven) from the second, showed an increase in interferon (IFN)-gamma production in response to allogeneic melanoma cell lines but not to controls. Four of five tested human leucocyte antigen (HLA)-A2(+) patients with anti-melanoma activity also showed specific T cell responses against peptides derived from melanoma-associated antigens. Delayed type IV hypersensitivity reaction (DTH) against melanoma cell lysate was observed in six of 13 patients from the group treated with DC vaccines only and four of seven from the group treated with the combination of DCs and IL-2. Significant correlations were found between DTH-positive responses against tumour lysate and both disease stability and post-vaccination survival on the stage IV patients. There were no toxicities associated with the vaccines or evidence of autoimmunity including vitiligo. Furthermore, no significant enhancement was observed as a result of combining DC vaccination with IL-2. Our data suggest that autologous DCs pulsed with tumour lysate may provide a standardized and widely applicable source of melanoma specific antigens for clinical use. It is safe and causes no significant side effects and has been demonstrated to be partially efficient at triggering effective anti-melanoma immunity.

Fig. 1

Phenotypic and functional properties of mature dendritic cells (DCs) at day 7. (a) Surface marker expression of mature DCs at day 7. Cells were treated as described in Materials and methods, marked with conjugated antibodies and analysed by flow cytometry. Only large cells corresponding to the forward scatter were selected for this evaluation. (b) Comparison of phagocytic capacity of immature and mature DCs. DCs from day 6 (immature) and day 7 [matured with tumour necrosis factor (TNF)-α] were mixed with fluorescein isothiocyanate (FITC)-conjugated dextran or with propidium iodide (PI)-labelled apoptotic melanoma cells as described in Materials and methods. After 2 h, cells were analysed by flow cytometry. Experiments were performed twice with similar results. (c) Cytokines released by matured DCs. Cells were incubated for 48 h in presence or absence of TNF-α, tumour cell lysate and allogeneic peripheral blood leucocytes (PBL) and the supernatants analysed by enzyme-linked immunosorbent assay (ELISA) as described in Materials and methods. The figure represents three experiments with DCs from different patients.

Fig. 2

Mature dendritic cells (DCs) loaded with melanoma cell lysate can stimulate interferon (IFN)-γ release by human leucocyte antigen (HLA)-A2 restricted melanoma-specific cytotoxic T lymphocytes (CTL) lines. Melanoma-specific T lymphocytes derived from (a) 0505 TIL or (b) DF CTL lines were incubated with DCs derived from melanoma patients (DC1 and DC2 are HLA-A2⁺ cells loaded with melanoma cell lysate; DC3 are HLA-A2⁺ cells loaded with a prostate cancer lysate; and DC4 are HLA-A2⁻ cells loaded with melanoma cell lysate) or K562 cells, as described in Material and methods and were then analysed by enzyme-linked immunosospot assay (ELISPOT), as described in Materials and methods. All ELISPOT experiments were performed in duplicate. Small graphs shows standard ⁵¹Cr release assays (a) 0505TIL and (b) DF CTL were incubated with ⁵¹Cr-labelled cells, as described in Material and methods.

Fig. 3

Interferon (IFN)-γ expression by melanoma specific T cells derived from peripheral blood mononuclear cells (PBMC) from responding patients. PBMCs from vaccinated patients CT001, CT003, CT006, CT007, CT010, CT011, CT013, CT014, CT015 and CT018 were obtained at different times and incubated with allogeneic melanoma cells, FM55mel, DFmel, FMSmel, DLmel or BEmel and with the natural killer (NK)-sensitive cell line K562 and were then analysed by enzyme-linked immunospot assay (ELISPOT), as described in Materials and methods. All ELISPOT experiments were performed in duplicate.

Fig. 4

Specific response of peripheral blood mononuclear cells (PBMC) from melanoma-vaccinated patients to melanoma-associated antigen. PBMC from HLA-A2⁺ patients were tested against FM55 melanoma cell line; T2 cells loaded with melanoma associated-antigen peptides: MART1/Melan A^27–35 (AAGIGILTV), gp100^280–288 (YLEPGPVTA) and tyrosinase^368–376 (YMDGTMSQV) or peptide HIV-1 p17 gag^77–85 (SLYNTVATL) and the natural killer (NK)-sensitive cell line K562 as controls and were then analysed by enzyme-linked immunospot assay (ELISPOT), as described in Materials and methods. The arbitrary units (aU) correspond to the number of spots obtained after vaccinations normalized with the amount of spots obtained from prevaccination peripheral blood leucocytes (PBL). All ELISPOT experiments were performed in duplicate.

Fig. 5

Immunological responses of vaccinated patients. (a) Typical DTH reaction to tumour cell lysate after two doses of dendritic cell (DC) vaccine. Similar reactions were detected in 11 out of 20 patients analysed. Correlation between delayed type hypersensitivity (DTH) positive response and (b) mean of time to progression (TTP) or (c) post- vaccination patient survival.