Ectopic expression of CCL19 impairs alloimmune response in mice

Abstract

Initiation of an antitumour immune response is characterized by a complex process of chemokine-mediated cell migration and cell-cell interactions. Overexpression of chemokine CCL19 in tumour cells has been shown to result in accelerated tumour rejection under certain experimental conditions, suggesting a novel approach in the therapy of neoplastic malignancies. To investigate CCL19-mediated modulations of cellular immune responses in vivo, we generated a chimeric CCL19-immunoglobulin G2b (IgG2b) Fc fusion protein, which binds to the chemokine receptor CCR7 comparable to native CCL19. CCL19-IgG2b possesses a long-lasting potent chemotactic activity as a result of the extended half-life of Fc fusion proteins. Stable overexpression of CCL19-IgG2b in BALB/c-derived J558L tumour cells fails to support tumour cell rejection following transplantation in syngeneic mice. Moreover, overexpression of CCL19-IgG2b hinders tumour rejection in allogeneic C57BL/6 mice. This phenomenon was accompanied by a six-fold increase of dendritic cells (DCs) isolated from CCL19-IgG2b-secreting tumours when compared to the number of DCs isolated from control parental J558L tumours. While mice bearing the allogeneic parental tumour showed an intense hypercellularity in the draining lymph nodes, no such response could be observed in the draining lymph nodes of mice carrying the CCL19-IgG2b-secreting tumour. We could demonstrate that overexpression of CCL19-IgG2b in tumour cells retains antigen-presenting cells in the tumour mass and prevents DCs from migrating into draining lymph nodes to present antigens and to activate T cells, resulting in an impaired immune response against the tumour.

Figure 1

Binding of CCL19-IgG2b to CCR7⁺ HUT78 cells and chemotactic activity. (a) Cells were incubated either with murine IgG (control) or CCL19-IgG2b (100 nm) followed by antimIgG-FITC-antibody. (b) Cells were preincubated with 100 nm recombinant CCL19 before staining with CCL19-IgG2b and anti-mIgG-FITC. (c) CCR7 is down-regulated by binding of CCL19-IgG2b in a temperature-dependent manner. FACS analysis of HUT78 cells after incubation with the CCL19-IgG2b fusion protein for the indicated times. Binding of fusion protein was detected by staining with α-murine IgG-FITC antibody. (d) Chemotactic responses of CCR7-expressing cells to rCCL19 and CCL19-IgG2b. HUT78 cells are stimulated with indicated concentrations of rCCL19, respectively, CCL19-IgG2b by using a 24-well Transwell chemotaxis chamber. The assay was done in triplicate. Shown is the percentage of migrated cells ± SD. (e) Effects of CCL19 on MAPK activation. Freshly isolated murine T cells were treated with rCCL19 or CCL19-IgG2b (10⁻⁷ and 10⁻⁶ m) at 37° for 5 min. The activity of MAPKs (ERK-2 and ERK-1 are indicated) was measured by specific phosphorylation. (f) As control for sample variations the blot was stripped according to the manufacturer's instructions (Amersham) and reprobed with p44/42 MAPK antibody, which detects total MAPK status independent of its phosphorylation, showing equal protein levels of ERK-2 and ERK-1.

Figure 2

(a) Constitutive secretion of CCL19-IgG2b does not suppress J558 tumour growth in syngeneic BALB/c mice. 5 × 10⁶ cells/200 μl PBS were injected s.c. in female 6–8-week-old BALB/c mice. The size of tumour was measured using a dial-gauge calliper (largest diameter × diameter at right angle). The groups differed not significantly regarding tumour size. Representative of three independent experiments, mean ± SD. (b) Immunohistochemically detection of CCL19 within the tumour of J558-CCL19-IgG2b-transfected cells documented intratumoral CCL19 expression in vitro. Indirect immunofluorescent staining of CCL19 with 10 μg/ml goat anti-mouse MIP-3β (R & D Systems, Wiesbaden, Germany) using peroxidase-conjugated mouse-anti-goat IgG (dianova) as secondary antibody and detection with tyramide signal amplification direct red (NEN Life Science Products, Boston, MA, USA), according to the manufacturer's instructions. Micrographs are representative of organs from three different recipient mice.

Figure 3

Increased tumour growth of CCL19-IgG2b-secreting J558 cells in allogeneic C57BL/6 mice; 5 × 10⁶ cells/200 μl PBS were injected s.c. in 6–8-week-old mice and tumour size was measured every day. Results are expressed as mean size ± SEM (representative of three independent experiments).

Figure 4

(a) CCL19-IgG2b-secreting J558 tumours suppress regional lymph node enlargement. Single cell suspensions of the presented lymph nodes were prepared and total cell numbers were counted. Data represent mean total cell numbers per lymph node ± SD (n = 5 mice per group). Numbers of mesenteric cells have been divided by 5. Experiment was performed in duplicate. (b) CCL19-IgG2b-secreting tumours decrease homing of lymphocytes into draining lymph nodes. Splenocytes from syngeneic donor animals were labelled with CFDA SE and injected intravenously into CCL19-IgG2b-secreting J558 tumour or parental J558 tumour-bearing mice on day 7, respectively. Representative results of independent experiments with five recipient animals each are shown. (c) increase of CD11b⁺CD11c⁺ DCs within the tumour mass of mice receiving CCL19-IgG2b-transfected tumour cells. Tumours of each group (untransfected J558 cells vs. J558-CCL19-IgG2b-secreting tumours) were harvested 7 days after tumour injection and analysed by FACS for the presence of infiltrated cells. (d) Distribution of infiltrated lymphocytes (CD3⁺ and B220⁺ cells) per mass of tumour. Comparison of parental J558 tumour cells vs. J558-CCL19-IgG2b-secreting tumour cells. Tumours of each group were harvested 7 days after tumour injection and analysed by FACS for the presence of infiltrated lymphocytes.