Preclinical exploration of combining plasmacytoid and myeloid dendritic cell vaccination with BRAF inhibition

Abstract

Background: Melanoma is the most lethal type of skin cancer and its incidence is progressively increasing. The introductions of immunotherapy and targeted therapies have tremendously improved the treatment of melanoma. Selective inhibition of BRAF by vemurafenib results in objective clinical responses in around 50 % of patients suffering from BRAFV600 mutated melanoma. However, drug resistance often results in hampering long-term tumor control. Alternatively, immunotherapy by vaccination with natural dendritic cells (nDCs) demonstrated long-term tumor control in a proportion of patients. We postulate that the rapid tumor debulking by vemurafenib can synergize the long-term tumor control of nDC vaccination to result in an effective treatment modality in a large proportion of patients. Here, we investigated the feasibility of this combination by analyzing the effect of vemurafenib on the functionality of nDCs.

Methods: Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were isolated from PBMCs obtained from buffy coats from healthy volunteers or vemurafenib-treated melanoma patients. Maturation of pDCs, mDCs and immature monocyte-derived DCs was induced by R848 in the presence or absence of vemurafenib and analyzed by FACS. Cytokine production and T cell proliferation induced by mature DCs were analyzed.

Results: Vemurafenib inhibited maturation and cytokine production of highly purified nDCs of healthy volunteers resulting in diminished allogeneic T cell proliferation. This deleterious effect of vemurafenib on nDC functionality was absent when total PBMCs were exposed to vemurafenib. In patients receiving vemurafenib, nDC functionality and T cell allostimulatory capacity were unaffected.

Conclusion: Although vemurafenib inhibited the functionality of purified nDC of healthy volunteers, this effect was not observed when nDCs were matured in the complete PBMC fraction. This might have been caused by increased vemurafenib uptake in absence of other cell types. In accordance, nDCs isolated from patients on active vemurafenib treatment showed no negative effects. In conclusion, our results pave the way for a combinatorial treatment strategy and, we propose that combining vemurafenib with nDC vaccination represent a powerful opportunity that deserves more investigation in the clinic.

Keywords: BDCA1 + myeloid DCs; DC vaccination; Plasmacytoid DCs; Vemurafenib.

Fig. 1

Vemurafenib bioavailability determines survival of pDCs and mDCs. Freshly isolated plasmacytoid DCs and myeloid DCs were cultured in increasing concentrations of vemurafenib and human serum and cell viability was determined after 24 h by FACS analysis

Fig. 2

Vemurafenib impairs maturation of ex vivo cultured pDCs and mDCs. Freshly isolated pDCs (a) and mDCs (b) were cultured ex vivo and activated with R848 in presence or absence of 60 μg/ml vemurafenib. Graphs show the cell surface expression levels of CD80, CD86, CD40, and PD-L1 after 18 h. Freshly isolated pDCs (c) and mDCs (d) were cultured ex vivo and activated with R848 in presence or absence of 60 μg/ml vemurafenib. Graphs show the levels of the cytokines IL-10, IL-6, TNFα and IFNα (only pDCs) measured in the supernatant after 18 h. Shown is the mean (+SEM) of six independent experiments *P < 0.05, **P < 0.01,***P < 0.001

Fig. 3

Dabrafenib and trametinib do not impair maturation of ex vivo cultured pDCs and mDCs. Freshly isolated pDCs (a) and mDCs (b) were cultured ex vivo and activated with R848 in presence or absence of 60 μg/ml vemurafenib, 90 ng/ml dabrafenib and 12 ng/ml trametinib. Graphs show the cell surface expression levels of CD80, CD86, PD-L1, MHC-I and MHC-II after 18 h. Shown is the mean (+SEM) of three independent experiments *P < 0.05, **P < 0.01,***P < 0.001

Fig. 4

Vemurafenib impairs antigen presentation and allostimulatory capacity of ex vivo cultured pDCs and mDCs. Freshly isolated pDCs (a) and mDCs (b) were cultured ex vivo and activated with R848 in presence or absence of 60 μg/ml vemurafenib. Graphs show the cell surface expression levels of MHC class I and MHC class II after 18 h. Mixed lymphocyte reaction using freshly isolated, pDCs (c) and mDCs (d) stimulated with R848 in presence or absence of 60 μg/ml vemurafenib. After maturation DCs were incubated with allogeneic PBLs and T cell proliferation was measured after 4 days with [³H] thymidine incorporation. d pDCs and mDCs from a HLA-A2.1 + donor were loaded with different concentrations of a melanoma-specific peptide (gp100_280:288) or irrelevant peptide (tyrosinase_369:376) in the presence of R848 with/without vemurafenib and gp100_280:288 specific T cells. Graphs show the cell surface expression levels of the early activation marker CD69 after overnight co-culture. Shown is the mean (+SEM) of at least three independent experiments *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

The presence of immune cells mitigates the inhibitory effects of vemurafenib on DC maturation and cytokine secretion. Freshly isolated PBMCs were cultured ex vivo and activated with R848 in presence or absence of 60 μg/ml vemurafenib. Graphs show the cell surface expression levels of CD80, CD86, CD40, and PD-L1 on pDCs (a) and mDCs (b) after 18 h. c Graphs show the levels of the cytokines IL-10, IL-6, TNFα, IL-1β, and IL-5 measured in the supernatant after 18 h. Shown is the mean (+SEM) of four independent experiments *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Vemurafenib does not inhibit ex vivo maturation of pDCs and mDCs isolated from melanoma patients on active treatment. PBMCs were isolated from patients before and after 1 month on vemurafenib treatment. a The frequency of pDCs and mDCs in the PBMCs was measured by FACS analysis. b Freshly isolated PBMCs from melanoma patients after 1 month on vemurafenib treatment were cultured ex vivo and activated with R848. Graphs show the cell surface expression levels of CD80, CD86, PD-L1, MHC-I, and MHC-II on pDCs and mDCs after 18 h. c Freshly isolated PBMCs from melanoma patients after 1 month on vemurafenib treatment were stimulated with PHA and T cell proliferation was measured after 4 days with [³H] thymidine incorporation. Shown is the mean (+SEM) of seven independent experiments *P < 0.05, **P < 0.01, ***P < 0.001