Intra-tumoral dendritic cells increase efficacy of peripheral vaccination by modulation of glioma microenvironment

Abstract

Pilot data showed that adding intratumoral (IT) injection of dendritic cells (DCs) prolongs survival of patients affected by glioblastoma multiforme (GBM) treated by subcutaneous (SC) delivery of DCs. Using a murine model resembling GBM, we investigated the immunological mechanisms underlying this effect. C57BL6/N mice received brain injections of GL261 glioma cells. Seven days later, mice were treated by 3 SC injections of DCs with or without 1 IT injection of DCs. DC maturation, induced by pulsing with GL261 lysates, was necessary to develop effective immune responses. IT injection of pulsed (pDC), but not unpulsed DCs (uDC), increased significantly the survival, either per se or in combination with SC-pDC (P < .001 vs controls). Mice treated by IT-pDC plus SC-pDC survived longer than mice treated by SC-pDC only (P = .03). Injected pDC were detectable in tumor parenchyma, but not in cervical lymph nodes. In gliomas injected with IT-pDC, CD8+ cells were significantly more abundant and Foxp3+ cells were significantly less abundant than in other groups. Using real-time polymerase chain reaction, we also found enhanced expression of IFN-gamma and TNF-alpha and decreased expression of transforming growth factor-beta (TGF-beta) and Foxp3 in mice treated with SC-pDC and IT-pDC. In vitro, pDC produced more TNF-alpha than uDC: addition of TNF-alpha to the medium decreased the proliferation of glioma cells. Overall, the results suggest that IT-pDC potentiates the anti-tumor immune response elicited by SC-pDC by pro-immune modulation of cytokines in the tumor microenvironment, decrease of Treg cells, and direct inhibition of tumor proliferation by TNF-alpha.

Fig. 1.

IT injection of pDC enhances anti-tumor efficacy of peripheral administration of DC. Kaplan–Meier survival curves represent the percentage of surviving animals over time (in days). Mice received 1 × 10⁵ GL261-glioma cells IT on day 0 and were treated with three SC injections of 1 × 10⁶ pDC spaced 1 week apart and/or one IT injection of 2 × 10⁵ GL261 Lysate-LPDC or UDC on day 7 after tumor implantation. The experimental conditions were: control mice received PBS (n = 15, reported in [A] and [B]); (A) IT-pDC (n = 10; P = .001 vs PBS); IT-uDC (n = 10; P = .02 vs PBS); IT-uDC plus SC-pDC (n = 10; P < .001 vs PBS); SC-uDC (n = 10, P = .2); (B) SC-pDC (n = 31, P < .0001 vs PBS); IT-pDC plus SC-pDC (n = 14; P < .0001 vs PBS). We divided the survival curves for clarity. Control curves are indeed the same. In Supplementary Material, Table S1, all P-values for treatment groups were reported vs control and vs each other group.

Fig. 2.

Intra-tumoral detection of pDC labeled by GFP or Endorem. DC-GFP can interact with tumor cells (A) and contact them with long spiky arms (B). Histological evaluations using Prussian Blue staining revealed iron in DC loaded with Endorem ex vivo 1 week after IT injection (C). Loaded DC can interact with tumor infiltrating, CD3+ lymphocytes (D).

Fig. 3.

Intra-tumoral levels of TGF-β, Foxp3, IFN-γ, and TNF-α. Tumors from control and treated mice were studied for the expression of TGF-β, Foxp3, IFN-γ, and TNF-α by RT–PCR at two different time points. Histograms show the evaluation of tumor environment 15 and 24 days after tumor implantation. (A) and (B) IT-pDC plus SC-pDC–treated mice TGF-β and Foxp3 expression is significantly lower than in SC-pDC–treated mice (TGF-β P = .009 and P = .01; Foxp3 P = .007 and P = .03, on day 15 and 24, respectively). (C) and (D) The same mice with decreased expression of TGF-β corresponded to higher expression of IFN-γ in IT-pDC plus SC-pDC–treated mice compared with SC-pDC–treated mice, particularly 24 days after tumor implantation (P = .03, 15 days; P = .002, 24 days). In contrast, on day 15, TNF-α expression was significantly higher in IT-pDC plus SC-pDC–treated mice than in SC-pDC–treated mice (P = .005, 15 days; P = .07, 24 days). Relative expression of cytokines in vaccinated mice was compared with controls.

Fig. 4.

Mature DCs produce “therapeutic” levels of TNF-α. For quantitative determination of cytokines produced by DCs, we tested the secretion of IL-6, TNF-α, and IFN-γ. The histograms in (A) show cytokine secretion by immature DCs (before pulsing) and mature DCs (24 hours after pulsing). Mature DCs (24 hours after pulsing) produce a higher level of TNF-α (mDC 343 pg/mL vs immDC 204 pg/mL); levels of IL-6 and IFN-γ were similar to immature DCs. (B) The histogram shows a significant decrease of cell proliferation observed after exposure to each concentration of TNF-α (black histogram, P < .0003). The exposure to IFN-γ decreased GL261 proliferation at higher concentrations (light grey histogram, P < .001) but not at 0.1 ng/ml (P = .2). The dark grey histograms show that GL261 were sensitive only to the highest concentration of IL-6 (100 ng/ml; P = .001).

Fig. 5.

Flow cytometric characterization of Treg from cervical lymph nodes. Foxp3+ cells from cervical lymph nodes show a time-related increase in control mice compared with vaccinated mice (from 10.0 ± 1.7% at 15 days to 19.5 ± 1.2% at 24 days, P = .001), reaching a maximum of 26.5 ± 4.9% at 31 days (P = .003 vs day 14; P = .03 vs day 24). In treated mice, the percentage of Treg decreases significantly when compared with controls (IT-pDC plus SC-pDC 6.3 ± 1.8, P = .0005; 9.4 ± 2.9, P = .04; 9.8 ± 1.3, P = .0004; SC-pDC 7.4 ± 0.6, P = .003; 10.6 ± 0.5, P = .01; 11.4 ± 1.2, P = .0007 on day 15, 24 and 31 respectively). In vaccinated mice, the percentage remains similar to healthy mice (9.4 ± 0.2%), but decreases slightly at 15 days. In Supplementary Material, Table S4, all P values for treated groups are reported vs controls and vs healthy mice.