In situ recruitment of antigen-presenting cells by intratumoral GM-CSF gene delivery

Abstract

Proper antigen presentation is paramount to the induction of effective and persistent antitumor immune responses. In a murine model of hepatic metastasis of colon cancer, we found that the numbers of in situ mature dendritic cells (DCs) and macrophages in tumor-infiltrating leukocytes (TILs) were significantly increased in mice treated with the combination therapy of herpes simplex virus thymidine kinase, interleukin 2, and GM-CSF genes when compared with control groups without GM-CSF treatment. Significantly higher levels of IFN-gamma, MIP-1 alpha, mIL-12, and GM-CSF were detected in the tumor after the combination therapy. T cells isolated from the combination therapy-treated mice exhibited higher ex vivo direct CTL activity than those from other treatment groups. Antigen-presenting cells (APCs) enriched from the TILs and liver of the combination therapy-treated mice induced higher levels of proliferation by the splenocytes from long-term surviving mice that had been cured of tumors at early time points (days 4 and 7) whereas significant APC activity was only observed in the spleen at the latter time point (day 7, 14) after the combination therapy. In contrast, APCs isolated from tk or tk + IL-2-treated mice did not induce any significant proliferation. Subcutaneous injection of fluorescence-labeled latex microspheres followed by the combination therapy showed a similar sequential trafficking of microspheres, day 4 after the combination therapy to tumor and day 14 to spleen. The results suggest that APCs recruited by intratumoral gene delivery of GM-CSF can capture antigens, mature to a stage suitable for antigen presentation, and subsequently migrate to the spleen where they can efficiently stimulate antigen-specific T cells.

Fig. 1.

Immunostaining of tumor-infiltrating leukocytes in hepatic colon tumors with dendritic cell–specific antibody (CD11c) (top images) and macrophage-specific antibody (F4/80) (bottom images) 2 days after various adenovirus treatments. The two left images are of tumor tissue from a tk + IL-2–treated animal. The two right images are of tumor tissue from a tk + GM-CSF + IL-2 treated animal. A significant increase in the numbers of DCs and macrophages infiltrating into the tk + GM-CSF + IL-2–treated tumor was observed as compared to the tk + IL-2–treated tumor (×400). No DC or macrophage in TK or control vector treated animals was observed (data not shown)

Fig. 2.

The phenotype of dendritic cells in the tumor from treated mice. Tumor-infiltrating leukocytes (TILs) were isolated and pooled from four mice per treatment group and stained for CD80 or MHCII (PE-labeled) in combination with CD11c (FITC-labeled). The result shown is a representative of three independent experiments

Fig. 3A–D.

Cytokine expression in the tumor tissues from mice receiving various virus treatments. Cytokine levels were measured following the freeze-and-thaw extraction method described in "Materials and methods," and the value was standardized by tumor weight. IL-12 levels are presented in A, MIP-1α in B, IFN-γ in C, and GM-CSF in D. The bars represent the mean of five mice tested and solid dots represent the values for individual animals. GM-CSF, MIP-1α, IFN-γ, and IL-12 levels were significantly higher in mice treated with tk + GM-CSF + IL-2 than in those from other treatment groups and the untreated tumor-bearing control group (P<0.05, one-way ANOVA test)

Fig. 4A, B.

Direct cytolytic T-cell response in the tumor-infiltrating leukocytes isolated from animals receiving various treatment combinations. A Treated mice receiving control Ig. The TILs derived from four mice in the various treatment groups were used for ex vivo cytotoxic assay. B Treated mice receiving MEL-14 Ab. The migration of naive T cells to lymph node was blocked by i.p. injection of MEL-14 antibody before and after the gene therapy. The standard error of the triplicate wells was less than 7%. Circles tk-treated group, squares tk + IL-2–treated group, triangles tk + IL-2 + GM-CSF–treated group

Fig. 5A–D.

Antigen presentation activity of leukocytes derived from the tumor, liver, or spleen of treated mice. Splenocytes from long-term surviving mice cured of tumors were used as the responder cells and enriched APCs from the tumor, liver, or spleen derived from various treatment groups, as stimulator cells. Freshly isolated splenocytes were cocultured for 5 days with irradiated APCs derived from different treatment groups at various time points after virus injection. Proliferation using a range of responder to stimulator (R/S) ratios was performed, but only the results from R/S = 1:0.08 are presented. ³H-Thymidine was added during the last 16 h of the culture. The results are presented as the relative proliferation index that is calculated by dividing the mean from triplicate of experimental group with that of control vector DL312 treatment group. A Antigen presentation activity in the tumor. B Antigen presentation activity in the liver. C Antigen presentation activity in the spleen. No significant antigen presentation activity was detected in the tumor, liver, and spleen of mice treated with tk or tk + IL-2 when compared with those treated with control vector DL312. The antigen presentation activity peaked on day 4 in the tumor and liver, on day 14 in the spleen of mice treated with tk + IL-2 + GM-CSF gene therapy. D The cell type responsible for APC activity. The enriched APCs were depleted of CD11c⁺ or F4/80⁺ cells and proliferation assay performed. Both dendritic cells (CD11c⁺) and macrophages (F4/80⁺) participated in the antigen presentation in mice treated with tk + IL-2 + GM-CSF gene therapy

Fig. 6A, B.

In vivo trafficking of APCs following intratumoral delivery of the GM-CSF gene. Fluorescent-labeled polystyrene microspheres were injected subcutaneously on the back of the mouse concurrent with virus injection into hepatic tumors. On days 2, 4, 7, and 14, hepatic tumors and spleens were isolated. After acetone extraction, as described in "Materials and methods," fluorescence intensity was measured using a fluorescence spectrophotometer. The results represent the mean of three individual animals. A The kinetics of fluorescence in the tumor of treated mice. B The level of fluorescence observed in the spleen of treated mice. The result shown is representative of three independent experiments. Circles fluorescence intensity from tk + GM-CSF + IL-2–treated mice, diamonds tk + IL-2–treated mice, squares untreated tumor-bearing mice