Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells

Abstract

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.

Figure 1

Immature DCs capture killed melanoma cells. (A) Colo829 cells are labelled with mAbs specific for gp100 (red) and Melan A/MART-1 (green). Confocal analysis shows that TAAs are expressed in different vesicular compartments. (B–D) DCs are cultured for 1 h with killed Colo829 cells and sorted according to size and granularity (forward/side scatter), fixed, and labelled with anti-gp100 (red) and anti–HLA-DR (green). Confocal microscopy analysis shows gp100 staining in the cytoplasm of most HLA-DR⁺ cells. (B) Projection of four xy serial sections. (C) Insert of a single section; original magnification: ×2.5. (D) xz vertical serial sections 1 2 3 and sum of gp100 staining of one cell 4. Representative of several experiments with Colo829 and Me275 cells. Bars, 10 μm.

Figure 2

DCs loaded with killed melanoma cells induce T cell proliferation. (a) Unloaded DCs (UL-DC) and loaded DCs (L-DC) are sorted and cultured with purified autologous CD4⁺ and (b) CD8⁺ T cells (with CD40L and IL-2). Thymidine incorporation at day 5. Representative of two experiments.

Figure 4

Induction of melanoma-specific CTLs by killed melanoma cell–loaded DCs. Purified CD8⁺ T cells are cultured for 3 wk with unloaded (UL-DC) or Me275-loaded DCs (L-DC). (a) IFN-γ ELISPOT using as targets DCs, either unpulsed or pulsed with the four melanoma peptides (4P). Representative experiment from three performed using DCs and T cells from different donors. (b) CTL activity in a 4-h chromium-release assay using unpulsed and melanoma peptide–pulsed (4P; mean values of three experiments) and control peptide PSA-pulsed T2 cells (one experiment).

Figure 3

Induction of CTLs by DCs loaded with killed melanoma cells. Purified CD8⁺ T cells are cultured for 3 wk with unloaded DCs (UL-DC) or DCs loaded with Me275 (L-DC). (a) T cells are tested in a 4-h chromium-release assay, using as targets immunizing Me275 cells and K562 cells as a control for NK activity (mean ± SE, n = 4), as well as (b) unrelated HLA-A201⁺ tumor cell lines, prostate cancer (LnCap) and breast cancer (1806). No CTLs are elicited when T cells are cultured with killed melanoma cells without the DCs (not shown).

Figure 5

DCs loaded with killed melanoma cells prime naive T cells. Naive CD8⁺CD45RA⁺CD45RO⁻ T cells are cultured for 3 wk with unloaded DCs (UL-DC) or with DCs loaded with killed melanoma cells (L-DC). T cells are tested in a 4-h chromium-release assay. (a) CTLs elicited by DCs loaded with Me275 bodies kill the T2 cells pulsed with different melanoma peptides (paired two-tailed t test on log transformed data comparing the killing of unpulsed and peptide-pulsed T2 cells). (b) Naive CD27⁺ CD45RA⁺CD8⁺ T cells differentiate into CTLs able to kill the cell line used for immunization, and (c) T2 cells pulsed with melanoma peptides (4P) but not PSA peptide (representative of two experiments).

Figure 6

Cross-priming using DCs loaded with killed melanoma cells. Naive CD27⁺CD45RA⁺CD8⁺ T cells are primed by autologous HLA-A201⁺ DCs loaded with killed HLA-A201⁻ Colo829 cells. The elicited T cells are able to kill HLA-A201⁺ melanoma targets but not HLA-A201⁺ breast cancer cells or autologous monocytes.

Figure 7

DCs loaded with killed melanoma cells stimulate melanoma-specific CD8⁺ T cells from the blood of patients with metastatic melanoma. Purified CD8⁺ T cells are cultured for 3 wk with unloaded (UL-DC) or Me275-loaded DCs (L-DC). (a) IFN-γ ELISPOT using DCs, either unpulsed or pulsed with four melanoma peptides (4P). Results from one patient. (b) T cells are able to kill Me275 used for immunization, and the killing is increased when Me275 cells are pulsed with the four melanoma peptides. Similar results were obtained in two patients. (c) CD8⁺CD45RA⁺CD45RO⁻ T cells cultured for 3 wk with DCs loaded with killed Me275 cells, kill T2 cells pulsed with four melanoma peptides.