Monophosphoryl lipid A plus IFNgamma maturation of dendritic cells induces antigen-specific CD8+ cytotoxic T cells with high cytolytic potential

Abstract

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNgamma generates mature DCs that produce IL-12 and polarize CD4(+) T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNgamma induce superior tumour antigen-specific CD8(+) CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNgamma DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNgamma DCs. The high amounts of induced CTLs are functional as they produce IFNgamma and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNgamma DCs are very promising for the use in future cancer immunotherapy.

Fig. 1

Phenotype, cytokine production, and chemokine expression of the matured monocyte-derived DCs. a DCs were harvested 2 days after maturation and stained for the different cell surface molecules with fluorescently conjugated mAbs and appropriate isotype controls. Histograms of the different markers are shown with the isotype matched controls as open graphs and the indicated marker Abs filled in grey. A representative experiment of eight experiments is shown. The mean fluorescent intensity ± SEM of all experiments in depicted in the upper right corner of the graphs. b IL-12p70, IL-23, IL-6 and IL-10 were determined in the culture supernatant 2 days after maturation. Harvested DCs were incubated with J558-cells expressing CD40L to determine the production of these cytokines upon CD40 restimulation. After 24 h the supernatant was harvested and cytokines were detected. The amount of cytokines produced by the two types of DCs from the same donor is connected by a line. A Wilcoxon matched pair t test was performed for statistical analysis; all cytokines produced were significantly different (P < 0.05) between the two DC types. c mRNA expression levels of CCL22 and CXCL10 were determined by real-time PCR 6 h after maturation. The expression of CCL22 in MPLA/IFNγ DCs was set to one. The expression of CXCL10 in CC-DCs was set to one. Relative mRNA expression (mean ± SEM) of four independent experiments is depicted

Fig. 2

DCs matured with MPLA/IFNγ are inducing high numbers of melanoma-antigen-specific CTLs. a 7 days after the second stimulation of the CTLs with DCs (day 17), the CTLs were stained with the MART-1 tetramer (TM) and CD8-FITC. A representative graph of CTLs stimulated with CC-DCs and MPLA/IFNγ DCs is shown. b A graph showing the % MART-1 tetramer cells from six separate cultures from one donor. A representative experiment out of seven experiments with independent donors is depicted. An unpaired t test was performed and the two DC conditions significantly differed (P < 0.0001). c In one donor the CTLs were not only stimulated with MART-1 peptide-loaded MPLA/IFNγ DCs, but also with gp100-209 peptide-loaded DCs. Before culture and after the second stimulation the CTLs were stained with CD8-FITC and MART-1 or gp100 tetramer, respectively. A graph showing the % tetramer positive CTLs from the separate cultures after two stimulations are shown. Before culture the donor was 0.11% MART-1 TM positive, gp100 TM positive cells were undetectable. d CTLs of melanoma patients were stimulated once with HLA-A2-matched DCs from a healthy donor. The MPLA/IFNγ DCs were loaded with MART-1, gp100-209 or gp100-280 peptide, respectively. Before and 1 week after stimulation the cells were analysed with the respective tetramers, a graph is showing the % TM positive cells at both time points

Fig. 3

CTLs, induced by MPLA/IFNγ DCs, are CD45RA⁻, CCR7⁻, CD28⁺ CD27⁺ and GrB⁺ and secrete IFNγ upon antigen-specific stimulation. a The phenotype of the CTLs were analysed 7 days after the third stimulation by staining the CTLs for MART-1 TM and analysing the expression of CD27, CD28, CCR7 and CD45RA on the MART-1 TM positive and negative CTLs. b The CTLs of the same cultures were incubated with JY cells loaded with either Her2 peptide or MART-1 peptide in an E:T ratio of 2:1 for 5 h in the presence of BFA (10 μg/ml). Subsequently, the intracellular accumulation of IFNγ was determined by FACS. CD8⁺ cells are depicted. c CD8⁺ cells were also analysed for the presence of intracellular GrB in combination with MART-1 TM staining. A representative CTL culture out of multiple cultures of two independent experiments is shown

Fig. 4

MART-1-specific CTLs, induced by MPLA/IFNγ DCs specifically and effectively lyse target cells. a ⁵¹Cr-loaded T2 cells either loaded with Her2 or MART-1 peptide were incubated in different ratio’s with CTLs in triplicate, 7 days after the third stimulation (day 24). Percentage of lysis by representative cultures from one experiment out of five experiments is shown. Mean ± SD of triplicates is depicted. MPLA/IFNγ DCs induced CTL culture A was 40% MART-1 TM positive and culture B was 20% MART-1 TM positive. CC-DC-induced CTL culture was 4.4% MART-1 TM positive. b The CTLs of the same cultures were also incubated with ⁵¹Cr-loaded MEL-AKR (MART-1+, HLA-A2+) and MEL-JKO (MART-1+, HLA-A2⁻) in different ratios. Percentage lysis is depicted as mean ± SD

Fig. 5

The superior induction of CTLs by MPLA/IFNγ DCs compared to CC-DCs is dependent on IL-12 and not on IL-6 or IL-23. a Blocking antibodies specific for IL-12p40 and IL-6, and an irrelevant antibody were added to MPLA/IFNγ DC-CTL cocultures. 7 days after the second stimulation of the CTLs with DCs (day 17), the CTLs were stained with the MART-1 tetramer (TM) and CD8-FITC. A graph showing the % MART-1 TM cells for all separate cultures of one experiment is shown. The condition treated with anti-IL-12 significantly differed (one-way ANOVA, P < 0.05) from untreated or isotype control treated conditions. A representative experiment out of four is depicted. b Blocking antibodies specific for IL-12p35 and IL-23p19, and an irrelevant antibody were added to MPLA/IFNγ DC-CTL cocultures. 7 days after the third stimulation of the CTLs, the CTLs were stained with the MART-1 TM and CD8-FITC. A graph showing the % MART-1 TM cells for all separate cultures of one experiment is shown. The condition treated with anti-IL-12p35 significantly differed (one-way ANOVA, P < 0.01) from isotype control treated condition