Dendritic cells infiltrating tumors cotransduced with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand genes take up and present endogenous tumor-associated antigens, and prime naive mice for a cytotoxic T lymphocyte response

Abstract

We transduced BALB/c-derived C-26 colon carcinoma cells with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (CD40L) genes to favor interaction of these cells with host dendritic cells (DCs) and, therefore, cross-priming. Cotransduced cells showed reduced tumorigenicity, and tumor take was followed by regression in some mice. In vivo tumors were heavily infiltrated with DCs that were isolated, phenotyped, and tested in vitro for stimulation of tumor-specific cytotoxic T lymphocytes (CTLs). BALB/c C-26 carcinoma cells express the endogenous murine leukemia virus (MuLV) env gene as a tumor-associated antigen. This antigen is shared among solid tumors of BALB/c and C57BL/6 mice and contains two epitopes, AH-1 and KSP, recognized in the context of major histocompatibility complex class I molecules H-2Ld and H-2K(b), respectively. DCs isolated from C-26/GM/CD40L tumors grown in (BALB/c x C57BL/6)F1 mice (H-2d x b) stimulated interferon gamma production by both anti-AH-1 and KSP CTLs, whereas tumor-infiltrating DCs (TIDCs) of BALB/c mice stimulated only anti-AH-1 CTLs. Furthermore, TIDCs primed naive mice for CTL activity as early as 2 d after injection into the footpad, whereas double-transduced tumor cells required at least 5 d for priming; this difference may reflect direct DC priming versus indirect tumor cell priming. Immunohistochemical staining indicated colocalization of DCs and apoptotic bodies in the tumors. These data indicate that DCs infiltrating tumors that produce GM-CSF and CD40L can capture cellular antigens, likely through uptake of apoptotic bodies, and mature in situ to a stage suitable for antigen presentation. Thus, tumor cell-based vaccines engineered to favor the interaction with host DCs can be considered.

Figure 1

In vivo tumorigenicity of C-26 cells and genetically modified variants. Cells (5 × 10⁴) were injected subcutaneously into the left flank of female BALB/c mice . ○, C-26; □, C-26/GM; •, C-26/CD40L; ▪, C-26/GM/CD40L. *P < 0.01; **P < 0.05, as determined using the Mann-Whitney test to compare transduced cell lines with the parental C-26 cells.

Figure 6

CTL activity after in vivo priming of BALB/c mice with CD11c⁺-enriched fraction of TIDCs (▪) or C-26/GM/CD40L control tumor cells (□). 10⁵ cells were injected in the footpad of naive BALB/c mice; after 5 (top panels) or 2 d (bottom panels), popliteal lymph nodes were removed. Lymphocytes were cultured in vitro with the AH-1–specific peptide and tested 6 d later for CTL activity.

Figure 2

FACS^® analysis of leukocyte infiltrate in tumors. (A) C-26/GM; (B) C-26/CD40L; (C) C-26/GM/CD40L. Tumor masses were surgically removed 15 d after implantation, then perfused with collagenase D solution, gently minced, and incubated in collagenase solution for 45–60 min at 37°C. After gentle pipetting and washing the suspension with DMEM several times, cells were seeded on 6-well plates at 0.7 × 10⁶ cells/ml and incubated overnight to allow tumor cell adherence to the plastic. Nonadherent cells were collected, double-labeled with CD8-PE/CD4-FITC (left) or CD11c-PE/B220-FITC (right), and analyzed by flow cytometry. Quadrants were set using isotype-matched antibodies as controls (not shown).

Figure 3

Phenotypic analysis of C-26/GM/CD40L TIDCs. DCs were purified using CD11c-conjugated microbeads. Sorted cells were double-stained for FACS^® analysis as follows: CD11c-PE/biotinylated MHC I (A), CD11c-PE/MHC II (B), CD11c-PE/B7.1 (C), and CD11c-PE/B7.2 (D). Unlabeled mAbs were detected with mouse-FITC. Isotype-matched antibody controls were used to set quadrants (not shown).

Figure 4

TIDC clusters after 3 d of in vitro culture on GM-CSF and CD40L cotransduced C-26 cells. After tumor mass digestion, the cellular suspension was seeded in 6-well plates at 0.7 × 10⁶ cells/ml; at day 3, nonadherent cells were collected and gently cytospun onto glass slides to avoid cluster disaggregation, then stained with Neat Stain kit (International PBI) (original magnification: ×400).

Figure 5

Apoptotic bodies within TIDCs. Double staining with DEC-205 and TUNEL reveals DCs engulfed with apoptotic bodies and blebs in C-26/GM/CD40L tumors (original magnifications: ×400; inserts: ×1,000).