Dendritic cells infected with poxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro

Abstract

Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful in enhancing the immunogenicity of tumor antigens and mediating tumor regression. Endogenous expression of antigen by DC offers the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. In this report, we show that human DC are (a) capable of infection by recombinant poxviruses encoding melanoma-associated antigen (MAA) genes and (b) capable of efficiently processing and presenting these MAA to cytotoxic T cells. In 6/6 HLA A*0201-expressing melanoma patients tested, the virally driven expression of MART-1/Melan A MAA by DC was sufficient to generate CD8+ T lymphocytes that could recognize naturally processed epitopes on tumor cells. In most cases, specific anti-MART-1 reactivity could be detected after a single stimulation. Analysis of epitope dominance revealed that the amino acid sequence recognized by these cytotoxic T lymphocytes (CTL) corresponded to the MART-1(27-35) residues previously shown to be most commonly recognized by cytotoxic T lymphocytes expanded from metastatic melanoma lesions. These data show that the virally driven expression of MAA by DC can be exploited for the efficient induction of clinically relevant cytotoxic T-cell responses. This has clinical implications for active immunization therapy, and currently vaccine trials have been proposed for patients with metastatic melanoma.

FIG. 1

Representative example of phenotype analysis of dendritic cells (DC) by fluorescence-activated cell sorter analysis. Surface phenotype of peripheral blood mononuclear cells (PBMC); adherent cells (AC) prepared on 75-cm² flasks after removal of nonadherent cells after a 3-h incubation with human recombinant interleukin-3 (hrIL-3) (200 IU/ml) at 37°C; and DC cultured with hrIL-4 (2,000 IU/ml) and human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) (2,000 IU/ml) at day 5. Cell fluorescence is indicated by solid histograms and the control staining with isotype-matched IgG in blank histograms.

FIG. 2

Morphologic appearance of dendritic cells (DC). Vital stain with calcein AM of DC after 5 days in culture with human recombinant interleukin-4 (hrIL-4) (2,000 IU/ml) and human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) (2,000 IU/ml) (A). Viral expression of MART-1 in the same DC by immunofluorescence (B). DC were incubated with rV-MART-1 for 1 h at 37°C at 10:1 multiples of infection. After 12 h, DC were stained with anti-MART-1 mAb (42). No staining or fluorescence was detected with anti-gp100 monoclonal antibody (mAb) (HMB45) as the negative controls. Conversely, DC infected with the rV-gp100 gene were not stained by anti-MART-1 mAb but were brightly stained with HMB 45.

FIG. 3

Recognition by the anti-MART-1_27-35 A42 cytotoxic T lymphocyte clone of endogenously processed MART-1 protein by rV-MART-1-infected dendritic cells (DC). Recognition is compared with that of MART-1_27-35 (1 μg/ml) pulsed DC. 10⁵ rV-MART-1 DC (black bars) or peptide-pulsed DC (hatched bars) from three melanoma patients were coincubated with 10⁵ A42 cells for 24 h at 37°C in 200 ml of culture medium. The efficacy of endogenous presentation by these DC was assessed by the induction of interferon-γ (IFN-γ) (pg/ml) release by A42. Negative stimulator cell controls included DC infected with an irrelevant rV encoding the sequence for the gp100 melanoma-associated antigens (white) or pulsed with the G9-209-2M peptide (gray bars). Similar results were obtained with rF-MART-1-infected DC.

FIG. 4

Specific interferon-γ (IFN-γ) release by CD8⁺ T lymphocytes from six HLA-A*0201 patients with metastatic melanoma. Anti-MART-1 cytotoxic T lymphocytes (CTL) were generated by stimulation with dendritic cells (DC) pulsed with MART-1_27-35 peptide or infected with rV-MART-1 and tested 7–9 days after stimulation (week 1). After 7 days, anti-MART-1 CTL stimulated with peptide-pulsed DC were restimulated with peptide-pulsed DC while cultures stimulated with rV-MART-1 DC were restimulated with DC infected with non-cross-reacting rF-MART-1. These CTL cultures were retested 7 days after the restimulation (week 2). Effectors were tested against T2 cells pulsed with 1 μg/ml peptide. Anti-MART-1 CTL cultures generated with peptide-pulsed DC were tested against T2 cells pulsed with 1 μg/ml of MART-1_27-35 (hatched bars) and G9-209-2M (gray bars) peptides. Anti-MART-1 CTL cultures generated with rV-MART-1 DC were tested against T2 cells pulsed with 1 μg/ml MART-1_27-35 (black bars) and G9-209-2M (white bars) peptides.

FIG. 5

Cytotoxic activity (calcein AM release) of anti-MART-1 cytotoxic T lymphocytes (CTL) induced with recombinant virus-infected dendritic cells (DC). Anti-MART-1 CTL were induced by a primary in vitro stimulation with DC infected with rV-MART-1 followed 7 days later with a second stimulation with DC infected with rF-MART-1 virus (rV-MART-1 = left two panels). By reversing the sequence of the viral vectors used for stimulation, anti-MART-1 CTL were also induced by a primary stimulation with DC infected with rF-MART-1 virus followed 7 days later by a second stimulation with DC infected with rV-MART-1 virus (rF-MART-1 = right two panels). Recognition of the epitope MART-1_27-35 was tested by pulsing T2 cells with 1 μg/ml MART-1_27-35 (■) or 1 μg/ml irrelevant G9-209-2M (□). Recognition of naturally processed MART-1 epitopes was tested against the 624.38 (▲) HLA-A*0201+/MART-1+ and the 624.28 (△), HLA-A*0201-/MART-1 + melanoma clones.