Human skin dendritic cells as target for anti-tumor vaccination Fehres, C.M.
2015
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citation for published version (APA)
Fehres, C. M. (2015). Human skin dendritic cells as target for anti-tumor vaccination.
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Unger WWJ, de Gruijl TD and van Kooyk Y .
1Department of Molecular Cell Biology and Immunology, VUmc Amsterdam, The Netherlands
2Department of Pathology, VUmc Amsterdam, The Netherlands
3Department of Medical Oncology, VUmc Amsterdam, The Netherlands
*Corresponding author.
European Journal of Immunology 2013 Aug 44;8:2415-24.
Chapter 6
Topical rather than intradermal application of the TLR7 ligand imiquimod leads to human dermal dendritic cell maturation and CD8
+T-cell cross-priming
6
Abstract
TLR ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by DCs. Given that human skin contains a dense network of DCs, which are easily accessible via (intra-)dermal delivery of vaccines, skin is actively explored as an anti-tumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara-treated DCs showed highest levels of the co-stimulatory molecules CD86, CD83, CD40 and CD70. Topical Aldara induced highest production of pro-inflammatory cytokines in skin biopsies.
When combined with intradermal peptide vaccination, Aldara-stimulated DCs showed enhanced cross-presentation of the melanoma antigen MART-1, which resulted in increased priming and activation of MART-1-specific CD8+ T cells. These results point to advantageous effects of combining the topical application of Aldara with anti-tumor peptide vaccination.
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Introduction
Vaccination is used to generate strong and specific immune responses against target antigens with minimal side effects. In particular, cancer vaccines aim to generate an efficient immune response against tumor-associated antigens (TAAs), which will depend on the instruction of CD8+ T cells that can effectively eradicate the tumor [1]. However, cancer vaccines often show poor immunogenicity, making them dependent on adjuvants to induce an immune response and to avoid the generation of tolerance.
Adjuvants consist of compounds that boost the potency, quality, or longevity of specific immune responses to antigens, but should cause minimal toxicity on their own [2]. They accomplish these effects via the generation of antigen depots, attraction of an immune infiltrate, enhancement of antigen presentation through the activation of antigen-presenting cells (APCs) including the induction of appropriate co-stimulatory molecules and cytokines [3]. Toll-like receptor (TLR) ligands have been extensively studied as the latest generation of adjuvants [4].
These receptors are widely expressed by both immune cells such as dendritic cells (DCs), and non-immune cells such as keratinocytes and fibroblasts [5]. TLRs represent a receptor family that recognizes a broad spectrum of conserved micriobial components [6]. For example, TLR7/8 recognizes viral ssRNA, but also small synthetic immune modulators, such as imiquimod (R837) and R848 [7-9].
Among cells of the hematopoietic lineage, TLR7 is mainly expressed by plasmacytoid and myeloid DCs as well as B cells [10;11]. TLR7-mediated activation of these cells resulted in the induction of the pro-inflammatory cytokines IFN-α, IFN-β, TNF-α and IL-12 [10]. In addition, enhanced migration, skewing to T helper type-1 (Th1) immune responses and an increased capacity for cross-presentation have been demonstrated after triggering of TLR7 on DCs [12]. For these reasons, the use of TLR7 agonists as adjuvant in cancer immunotherapy is actively being explored.
However, systemic administration of TLR7 agonists has been associated with chronic immune activation, inflammation and systemic endothelial activation, raising questions concerning safety and drug toxicity [13].
Imiquimod 5% cream (Aldara; Graceway Pharmaceuticals, LLC, Exton, PAA) is an FDA- approved immune response modifier available for the treatment of genital warts, actinic keratinosis and superficial basal cell carcinoma [14]. Previous studies have shown that the topical application of Aldara skin cream results in increased levels of Th1 cytokines, activation of NK cells and enhanced antigen presentation by APCs, without causing considerable side effects in humans [15]. Moreover, repeated topical application of only Aldara on the shoulders and flanks improved the survival of mice bearing intracranial glioma and breast tumors, and was associated with elevated numbers of tumor-infiltrating CD4+ and CD8+ T lymphocytes [16]. These findings indicate that local application of imiquimod via the skin may represent a good strategy to modulate systemic immunity and to induce anti-tumor effects. Currently,
6
it is not yet clear what the effects of Aldara skin cream are on human skin DCs.
Skin has increasingly been used as a site for vaccination, mainly because of its high prevalence of APCs with immunostimulatory and migratory capacities [17]. Steady- state human skin contains at least three phenotypically and functionally distinct subsets of DCs: CD1ahigh Langerhans cells (LCs) reside in the epidermis, whereas CD1a+ (also known as CD1c+ DCs) and CD14+ DCs are present within the dermis[18-23].
It has been shown that LCs are superior in the priming and cross-priming of CD8+ T cells, whereas CD14+ dermal DCs (dDCs) are able to induce the generation of follicular T helper cells [18;24]. CD1c+/CD1a+ dDCs are recently described as the functional equivalents of mouse CD11b+ DCs and show Th17 polarizing capacities [25]. Additionally, it has been shown that human CD1a+ dDCs are capable to stimulate CD4+ T-cell proliferation as well as to prime tumor-specific CD8+ T cells [19;26]. Furthermore, LCs are less responsive than the dermal DC subsets to bacteria due to a lack of TLR2, TLR4 and TLR5 expression [19;27;28]. CD14+ dDCs express most of the ten human TLRs, while CD1a+ dDCs seem to express all TLRs with exception of TLR9 and TLR10 [19;27;28].
We previously showed that intradermal delivery of TLR ligands in a human skin explant model had modest effects in terms of skin DC maturation with enhanced T-cell stimulation only by the TRL2 ligand PGN and the TLR3 ligand polyI:C [29]. In this study we have investigated the effects of epicutaneous application of imiquimod in comparison with the intradermal injection of imiquimod or R848, which is also a TLR7/8 ligand. The difference in application might trigger other skin cells and consequently lead to differences in functional outcome. Soluble TLR ligands were injected intradermally into healthy human skin explants and Aldara skin cream was topically applied. This set-up is highly representative of the physiological 3D situation. Our data show that application of Aldara skin cream results in increased numbers of migrated dermal DCs as compared with that after the intradermal injection of soluble TLR ligands, including imiquimod. Additionally, Aldara-treated skin DCs were highly mature and induced superior activation of CD8+ T cells specific for the melanoma antigen MART-1. These results provide evidence that Aldara skin cream is a suitable adjuvant to combine with intradermal peptide vaccination in order to induce anti-tumor immune responses.
Results
Effects of TLR ligands on skin resident DC migration
In order to analyze the effects of Aldara skin cream or the soluble ligands for TLR3, TLR7 and TLR8 on skin resident DCs, we first evaluated the effects of the stimuli on DC emigration. Human skin explants were intradermally (i.d.) injected with the soluble TLR ligands or the cytokines GM-CSF/IL-4 and Aldara skin cream was applied topically. Intradermal injection of a volume of 20 μl did not affect the skin morphology
6
or disturbed the basal membrane between epidermis and dermis, as show in Supporting Information Figure 1. We included GM-CSF/IL-4 as a positive control, since these cytokines were earlier described to be potent inducers of skin DC maturation and emigration[30]. Aldara-treated skin was also injected with 20 μl of IMDM/biopsy to exclude differences in DC migration and maturation due to tissue rupture caused by the injections. Numbers and percentages of DCs determined by Forward and Side Scatter properties and the expression of HLA-DR were measured in the total population of cells that emigrated from the skin biopsies (Fig.1A-B).
Injection of IMDM alone resulted in the migration of an average of 4000 HLA-DR+ DCs after 2 days of culture. By contrast, injection of the cytokines GM-CSF/IL-4 or
1 A B
CD1a
SSC
FSC HLA-DR
FSC
CD14
100101102 103104 100
101 102 103
10433 12
24 31
100 101 102 103 104
76.2 0.8
21.4 1.6 GM/4 IMDM
C D
100 101 102 103 104 100
101 102 103
10479.5 7.2
Langerin 0 200 400 600 800 1000
100 101 102 103 104
97.3
0 200 400 600 800 1000 0
200 400 600 800 1000
19.5
GM/4 100 101 102 103 104 IMDM GM/4
R848 R837
Aldara 0
2500 5000 7500 10000
12500 *** **
# of migrated HLA-DR
+ cells measured in 1 minute of aquisition
CD14 dDCs
IMDM GM/4
R848 R837
Aldara 0
5 10 15 20 25 30 35 ***
% CD14+HLA-DR+ DCs
CD1a dDCs
IMDM GM/4 R848
R837 Aldara 0
10 20 30 40 50 60 70 80 ***
IMDM GM/4
R848 R837
Aldara 0
2 4 6 8 10 12 ***
% HLA-DR
+ Langerin+ cells CD1a
% CD1a+HLA-DR+ DCs
1. Intradermal injection of TLR ligands does not influence the migration of DCs from human skin biopsies.
After i.d. injection of the indicated TLR ligands or application of Aldara skin cream, 6-mm biopsies per condition were taken immediately and cultured for 2 days. Migrated cells were pooled per condition and stained for HLA-DR. (A) The absolute numbers of migrated DCs out of 8 pooled biopsies per condition were identified based on the FSC/SSC properties and staining for HLA-DR. Flow cytometer acquisition was set on 1 minute and the numbers of migrated DCs were determined based on the number of HLA-DR+ cells per condition. Data are shown as mean + SEM pooled from 5 donors. The amount of migrated DCs out of 8 pooled biopsies per condition was determined, measured as a single observation per donor. (B) Gating strategy to identify CD14+ and CD1a+ dDCs and CD1a+Langerin+ LCs after migration. (C) Percentages of migrated CD14+ and CD1a+ dDCs were determined within the population of migrated HLA-DR+ cells by flow cytometry. Data are shown as mean + SEM from 5 skin donors. For each donor, the amount of migrated CD1a+ and CD14+ dDCs out of 8 pooled biopsies per condition was determined, measured as a single observation per donor. (D) Percentages of migrated CD1a+ Langerin+ LCs were determined within the population of migrated HLA-DR+ cells. Data are shown as mean + SEM from 3 donors. For each donor, the percentage of LCs was determined on migrated cells pooled from 8 biopsies per condition. Statistical significance was determined using an ANOVA combined with a Bonferroni Multiple Comparison test;
**p<0.01 and ***p<0.001.
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application of the imiquimod-containing Aldara cream resulted in significantly higher absolute numbers of migrated DCs (Fig.1A).However, injection of the soluble TLR ligand R848 (TLR8) or the soluble form of imiquimod (R837; TLR7 ligand) did not result in an upregulation of migratory DC numbers. Next, we analyzed whether these stimuli differentially affected the migratory potential of the various skin resident DC subsets. To this end, migrated cells were stained for HLA-DR, CD1a, CD14 and Langerin, enabling the discrimination between two dermal DC subsets and LCs (fig. 1B). As shown in Figure 1B and C, the percentages of migrated CD14+ and CD1a+ dDCs of all HLA-DR+ dDCs were equivalent between the human skin biopsies injected with IMDM, R848 or R837 or topically treated with Aldara. Only i.d. injection of GM-CSF/IL-4 caused a shift in migrated dDC subsets, namely an almost 8 fold decrease in CD14+ dDCs and a 2.2 fold increase in the percentage of migrated CD1a+ dDCs compared to injection of IMDM, which is equivalent to our previous observations[30]. In addition, as shown in Fig.1D, only injection of GM- CSF/IL-4 led to significantly enhanced migration of LCs (almost 6 fold increase compared to IMDM). Neither injection of the soluble TLR7 and TLR8 ligands, nor the application of Aldara cream did affect the migration of LCs compared to IMDM.
Epicutaneous Aldara treatment leads to phenotypical maturation of skin DC subsets Next, we determined the effects of the differently delivered TLR ligands on DC maturation. Migrated DCs were stained for CD14 and CD1a in order to study the effects of TLR stimulation on the dDC subset maturation status individually. As shown in Figure 2A, injection of R848 and even R837 did not result in a significant higher expression of CD86, CD83 and CD70 as compared to IMDM treated CD14+ and CD1a+ dDC. In contrast, epicutaneous Aldara treatment did result in a significant higher expression of CD86 and CD70 on the CD1a+ dDCs, indicating that Aldara treatment most efficiently induced dDC maturation.
Since CD70 is of importance for mature DCs to activate and prime CD8+ T cells, we analyzed the expression of CD70 in more detail on the 3 main skin DC subsets after intradermal injection of IMDM or R837 or after topical application of Aldara skin cream. Although i.d injected R837 induced a higher percentage of CD70+ cells in all subsets compared to IMDM, application of Aldara skin cream induced even a higher percentage of CD70+ LCs, CD1a+ dDCs and CD14+ dDCs (Fig. 2B).
In addition, we compared the effects of the intradermal injections of GM-CSF/IL-4 and R837 with the epicutaneous application of Aldara on the DC maturation profile in more detail (Fig. 2C). Epicutaneous Aldara treatment resulted in a more matured phenotype of the migrated dDCs, with significantly increased levels of MHC class I, CD86, CD40 and CD70 (Fig.2C) compared to IMDM (all markers) and GM-CSF/IL-4 (CD70 and CD86). In contrast, intradermal injection of R837 did not result in a significant upregulation of any of the maturation markers compared to IMDM or GM-CSF/IL-4. Together, these results demonstrate that Aldara skin cream treatment results in migration of high numbers of phenotypically mature skin dDCs.
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2 A CD70 CD83 CD86
CD14+DCs
CD1a+DCs
C
0 400 800 1200 1600 2000 2400
0 50 100 150
0 400 800 1200 1600 2000
2400 * *
0 50 100 150 0
25 50 75 100
MFI
25 50 75
100 *
MFI
CD70 CD83 MHC I CD40 CD86
0 30 60 90
0 500 1000 1500
2000 *
*
0 25 50 75
100 **
***
MFI
0 50 100 150 200
250 *
0 200 400
600 *
B
0IMDM GM/4 R848 R837 Aldara IMDM GM/4 R848 R837 Aldara
0IMDM GM/4 R848 R837 Aldara
2. Expression of activation markers on skin migrated DCs. (A) Expression of activation markers on CD14+ and CD1a+ migrated dDCs 48 hours after injection of IMDM, GM-CSF/IL-4, R848 and R837 or application of Aldara skin cream. The MFI of each marker is shown as mean + SEM of at least 3 donors per condition. (B) Dot plots of the CD70 expression on the main human subsets from one representative experiment are depicted. Numbers in each quadrant represent percentages. (C) Comparison of the expression levels of various activation markers on migrated DCs after i.d. injection of IMDM, GM-CSF/IL-4 or R837 or epicutaneous application of Aldara, as determined by flow cytometry. Data are shown as mean + SEM of 5 individual donors. Statistical significance was determined using an ANOVA combined with a Bonferroni Multiple Comparison test; *p<0.05, **p<0.01 and ***p<0.001.
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Elevated levels of pro-inflammatory cytokines produced by skin explants after Aldara treatment
Besides phenotypic maturation, we were also interested in the potency of imiquimod, either after i.d. injection or topical application to human skin, to stimulate the production of cytokines in the skin microenvironment. Supernatants of the cultured biopsies were taken 2 days after i.d. injections or Aldara application and amounts of IL-6, IL-8, TNF-α, IL-1β and IL-10 were determined by ELISA. Compared to injection of medium only, injection of GM-CSF/IL-4 resulted in a 2-fold higher expression of TNF-α (Fig. 3A).However, application of Aldara cream to the skin resulted in significantly higher levels of TNF-α (2-fold increase), IL-1β (approximately 10-fold increase), IL-10 (2-fold increase), IL-6 (3-fold increase) and IL-8 (approximately 10-fold increase)produced by the skin biopsies when compared to IMDM. Remarkably, i.d. injection of the active component of Aldara skin cream, R837, did not result in any significant increase in the production of pro-inflammatory cytokines.
IMDMGM/4 R837
Aldara 0
10 20 30
40 *
TNF-α (pg/ml)
IMDMGM/4 R837
Aldara 0
50 100
150 ******
**
IL-6 (ng/ml)
3 A IL-6 IL-8
TNF-α IL-10
IL-1β
B
IMDM GM/4
R837 Aldara 0
30 60 90 120
150 * ***
*
IL-10 (pg/ml)
IMDMGM/4 R837
Aldara 0
10 20 30 40 50 60
70 **
**
IMDM GM/4 R837
Aldara 0
40 80 120
******
***
IL-1β (pg/ml)
IMDM GM/4 R837
Aldara 0.0
0.2 0.4 0.6
0.8 * **
rel. expression to GAPDH
IL-1β mRNA produced by KCs
IL-8 (ng/ml)
3. Application of Aldara on the skin induced the production of cytokines in the cultured skin biopsies. (A) Biopsies were either i.d. injected with IMDM, GM-CSF/IL-4 or R837 or Aldara cream was applied topically and cultured for 48 hours, after which conditioned medium was harvested and analyzed for the presence of the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α by ELISA. Data are shown as mean + SEM from at least 3 skin donors. (B) IL-1 mRNA expression by human keratinocytes isolated after Aldara treatment was measured by RT-PCR. Data are shown as mean + SEM of 1 experiment performed in quadruplicate. *p<0.05, **p<0.01 and
***p<0.001. Statistical significance was determined using an ANOVA combined with a Bonferroni Multiple Comparison test.
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Since keratinocytes are the main cells present the epidermis, we hypothesized that they might be important for the production of inflammatory cytokines as a consequence of the atopically applied Aldara cream. In order to study the possible role of keratinocytes in the production of TNF-α and IL-1β upon Aldara treatment, keratinocytes were isolated from the epidermis of differently treated skin biopsies.
As shown in Figure 3B, keratinocytes treated with Aldara produced a 3 times higher level of IL-1β mRNA compared to those treated with IMDM or GM-CSF/IL-4.
Surprisingly, keratinocytes derived from biopsies intradermally injected with R837 showed comparably high levels of IL-1β mRNA to the Aldara-treated keratinocytes (Fig.3B). Furthermore, keratinocytes derived from biopsies treated with GM-CSF/
IL-4, R837 or Aldara skin cream did not show any increase in TNF-α mRNA levels compared to IMDM alone, suggesting that TNF-α is produced by other cells in the skin (data not shown).
Induction of tumor-specific CD8+ T cells by DCs derived from Aldara-treated skin biopsies
To study the effects of Aldara on the capacity of skin DCs to process and present peptide antigen to CD8+ cytotoxic T cells, biopsies were i.d. injected with a 15 amino acid long MART-1 peptide containing the MHC class I epitope. Migrated DCs derived from explants injected with 10 μg/biopsy MART-1 peptide in combination with IMDM, GM-CSF/IL-4 or R837 or Aldara application were co-cultured with a MART-1- specific CD8+ T cell clone for 24 hours, after which supernatants were analyzed for the presence of IFN-γ. As depicted in Figure 4A, only the Aldara-treated skin DCs showed an enhanced ability to cross-present the MART-1 peptide, resulting in significant more production of IFN- γ by the CD8+ T cells. As shown in Figure 4B, the 15 AA long MART-1 peptide needs to be processed by DCs, since it cannot bind exogenously in MHC class I molecules and directly induce activation of the MART-1- specific CD8+ T cell clone, whereas the minimal MART-126-35 epitope, that serves as a positive control, resulted in high IFN-γ production by the T cells. Furthermore, we analyzed the CD8+ T cell response after injection of titrated concentrations of MART-121-35 peptide in skin treated with i.d injection of IMDM and GM-CSF/IL-4 or with application of Aldara. Figure 4C also demonstrates superior processing and presentation of the MART-121-35 peptide by DCs from Aldara-treated skin explants as compared to DCs from GM-CSF/IL-4- or IMDM-injected skin explants. To determine whether DCs derived from Aldara-treated skin also showed superior induction of MART-1-specific, naïve CD8+ T cells, we repeated the antigen presentation assays using HLA-A2+ DCs migrated from GM-CSF/IL-4-injected and Aldara-treated explants and co-cultured them in a series of experiments with primary CD8+ T cells isolated from blood from HLA-A2 matched donors. Figure 4D shows representative results demonstrating that DCs derived from Aldara-treated skin induced an increased outgrowth of MART-1 tetramer positive CD8+ T cells as compared to GM-CSF/IL-4 modulated DCs. In addition, the percentages of CD8+ T cells primed by DCs derived
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01 2 3 4
5 GM/4
Aldara
*
**
% IFN-γ+
0 1.25 2.5 5 10
0 100 200
300 IMDM
GM/4Aldara
**
*
µg MART-121-35/biopsy
B C
D
0 1 3 9
0.0 1.5 3.0 4.5 GM/4
Aldara
% Tm+ CD8+ T cells
** Aldara F
4 A
26-35
MART-1 21-35 MART-1 none 0
100 200 300 400
IFN-γ (pg/ml)
TNF-α
IFN-γ
E GM/4
0.3 3.3 0.93
100 104 103 102 101
0.1 1.0 0.46
100 101 102 103 104101000 101 102 103 104 104
103 102 101 0
2 4
6 *
Fold increase in IFN-γ
IMDM GM/4 R837 Aldara
IFN-γ (pg/ml) CD8+ T cells
0 1 3 9 µg MART-121-35/biopsy
4. Increased antigen presentation to MART-1-specific CD8+ T cells by Aldara-treated DCs. (A) DCs migrated from skin biopsies injected with 10 μg/biopsy of MART-121-35 peptide in combination with IMDM, GM-CSF/
IL-4, R837 or application of Aldara skin cream were co-cultured with MART-1-specific CD8+ T cells. After 24 h of co-culture, supernatants were harvested and IFN-γ levels were analyzed using ELISA. The fold increase in IFN-γ levels is depicted with the IFN-γ levels produced in the IMDM condition set at 1. DCs derived from Aldara-treated skin consistently induced highest levels of IFN-γ produced by the CD8+ T cells. IFN-γ levels ranged from 30 pg/ml to 380 pg/ml. Combined data of 5 separate experiments are shown as mean + SEM, each sample measured in triplicate. (B) MART-1-specific CD8+ T cells were incubated with 10 μg/ml of purified 10 AA-long MART-126-35 or with the 15 AA-long MART-121-35 peptide. After 24 h of stimulation, supernatants were harvested and IFN-γ levels were analyzed using ELISA. Data are shown as mean + SEM from 3 individual experiments and samples were measured in duplicate. (C) DCs derived from skin biopsies injected with indicated concentrations of the purified MART-121-35 peptide in combination with IMDM, GM- CSF/IL-4 or application of Aldara skin cream were co-cultured with MART-1-specific CD8+ T cells and IFN-γ levels were analyzed of 4 samples pooled from 2 experiments. (D) Increased antigen presentation to MART- 1-responsive primary CD8+ T cells from allogeneic donors by Aldara-treated DCs. T cells were expanded for 7 days and analyzed using flow cytometry to determine the percentage of MART-1 tetramer-specific CD8+ T cells. Combined data are shown as mean + SEM of 3 donors, each sample measured in duplicate. (E, F) DCs migrated from skin explants which were injected with (E) 9 μg of MART-1 or (F) the indicated concentrations of MART-1 were co-cultured for 7 days with primary CD8+ T cells. CD8+ T cells were then stimulated either with i.d. injected GM-CSF/IL-4 or Aldara cream was applied topically. TNF-α and IFN-γ production was determined by flow cytometry. (E) A representative experiment (F) and the combined data of 2 experiments, each performed in duplicate, are shown. Mean ± SEM of 2 samples pooled from 2 experiments. Statistical significance was determined using an one-way ANOVA combined with a Bonferroni Multiple Comparison test for Figure 4A and a two-way ANOVA combined with a Bonferroni Multiple Comparison test for Figure 4C,D and E; *p<0.05 and **p<0.01.
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from Aldara-treated skin producing intracellular IFN-γ and TNF-α were also significantly higher compared to the percentages of IFN-γ- and TNF-α-producing T cells primed by GM-CSF/IL-4 stimulated DCs (Fig. 4 E+F).
Discussion
Here, we show that application of the TLR7 ligand R837 containing Aldara skin cream has superior potential as adjuvant for human skin-based vaccination protocols. Application of Aldara skin cream resulted in enhanced migration of CD1c+/CD1a+ and CD14+ dermal DCs as compared to i.d. injection of the soluble form of the active component of Aldara, the TLR7 ligand R837. In addition, Aldara- treated DCs showed an enhanced maturation profile both phenotypically as well as on the level of cytokine production. More importantly, Aldara cream resulted in DCs that were superior in the activation of melanoma antigen MART-1-specific CD8+ T cell clone and induction of antigen-specific, IFN-γ and TNF-α producing effector CD8+ T cells in primary T cell responses.
Out of a panel of soluble TLR agonists or stimulatory cytokines, only the TLR7 and TLR8 agonist present in Aldara cream consistently induced phenotypic activation and enhanced migration by skin DCs. The potential function of Aldara skin cream as vaccine adjuvant is also shown in a patient study where the safety and feasibility of Aldara as adjuvant in a vaccine against the cancer/testis antigen NY-ESO-1 and the immunogenicity of the combination was evaluated in malignant melanoma patients[31]. The combination of protein vaccination and Aldara as adjuvant was well tolerated and elicited both humoral and cellular responses in a significant fraction of patients, demonstrating the feasibility of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients.
Strikingly, only the topical application of R837 in the form of Aldara resulted in enhanced activation and migration of dDCs, whereas i.d. injection of soluble imiquimod (R837) did not show these responses. This might be explained by the difference in mode of application: Aldara skin cream is directly applied on the epidermis, where also the keratinocytes reside. In contrast, the soluble TLR ligands were injected into the dermis of the skin, thus by-passing the epidermis. Human keratinocytes constitutively synthesize pro-IL-1β, but do not secrete the processed, activated form under normal conditions [32]. However, upon stimulation of the skin with immunostimulants (like Aldara skin cream), keratinocytes secrete the active form of IL-1 β, which in turn has immune stimulatory effects on many cell types present in the skin tissue microenvironment, most notably setting in motion a chain of events leading to DC activation and migration [32;33]. Another explanation might be the composition of the cream. One of the most commonly reported side effects of Aldara treatment is local erythema at the application site [34;35]. It has been reported that patients treated with placebo cream also developed erythema and
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itching, indicating that the cream itself already has activating effects on the skin microenvironment [36]. Recently, Walter et al. provided evidence that isostearic acid, a major component of the cream, promotes inflammasome activation in cultured keratinocytes [37]. Thus, the effects of the cream itself might contribute to the enhanced DC stimulatory effects of Aldara as compared to i.d. injections of soluble R837.Furthermore, although we adjusted the concentration of soluble R837 injected i.d. to the concentration of R837 in the Aldara skin cream that was spread on the skin, we can not truly rule out that the our observed results are in part due to a difference in the effective concentrations of R837 between the cream and soluble R837.
Human skin contains 3 main subsets of DCs: epidermal Langerhans cells and CD14+
and CD1a+ DCs present in the dermis [18;20;21;23]. In recent years, a lot of effort has been put in identifying the contribution of each subset to the induction of immunity or tolerance. It has been reported that LCs show specialization for induction of cytotoxic CD8+ T cells, whereas CD14+ dDCS are specialized in inducing antibody production and isotype switching of B cells[18]. Unexpectedly, administration of GM-CSF/IL-4 did not result in the highest capacity of migrated DCs to activate and prime CD8+ T cells, although GM-CSF/IL-4 has been shown to cause enhanced migration of the CD8+ T cell priming-subsets LCs and CD1a+ dDCs (Fig.1).
Also, i.d. injections of GM-CSF/IL-4 reduced the percentage of migrated CD14+ dDCs, which are not the DCs known for CD8+ T cell activation (Fig.1). Moreover, we show that DCs migrated from Aldara-treated explants have superior potential to activate naïve CD8+ T cells to a higher extent than DCs migrated from biopsies injected with GM-CSF/IL-4. These effects could be possibly explained by the upregulation of CD40 and CD70 on Aldara-treated skin DCs compared to GM-CSF/IL-4 stimulated DCs, since both molecules are described to be important in CD8+ T cell stimulation by DCs (Fig.2B). Expression of CD70 on DCs is reported to favor the priming of CD8+ T cell responses and the generation of CD8+ T cell memory [38;39], whereas CD40 engagement on DCs promotes cytokine production, induces co-stimulatory molecules on the surface of the DCs and facilitates cross-presentation of antigen [40]. Recently, a paper Nizolli et al. described the enhanced capacity of human CD1c+ DCs to cross-present soluble antigens after simultaneous stimulation with the TLR4 ligand LPS and the TLR8 ligand R848 [41]. These results indicate that TLR7/8 agonists, like Aldara skin cream, R837 and R848, might have positive effects on the capacity of CD1c+ DCs to cross-present. In line with this, our study has shown that the topical application of Aldara skin cream has superior effects on skin DC cross- presentation compared to IMDM or GM-CSF/IL-4. However, we could not find evidence that the soluble TLR ligands R837 or R848 would have a positive effect on antigen cross-presentation by CD1a+ skin DCs, since intradermal injection of R837 or R848 did not induce the increased expression of co-stimulatory molecules or the production of inflammatory cytokines by CD1a+ dDCs. These differences might be explained by the models tested (in vitro stimulation of blood or tonsil derived CD1c+
6
DCs versus the stimulation of DC subsets within the skin micro-milieu) or the mode of adjuvant delivery (intradermal injections versus topical application of R837).
Altogether, Aldara skin cream is an attractive candidate adjuvant in combination with intradermal delivery of a therapeutic vaccine against melanoma. Aldara skin cream resulted in increased numbers of migrated cutaneous DCs and led to an enhanced maturational status of the migrated DCs. Additionally, DCs migrated from biopsies treated with Aldara were superior in the priming and activation of MART-1-specific CD8+ T cells. Therefore, the combined administration of anti-tumor peptide vaccines and Aldara as adjuvant should be further explored as treatment of skin malignancies.
Material and methods Reagents
Aldara skin cream was purchased from Meda AB (Solna, Sweden) and stored at room temperature. For each experiment 1 sachet was used. rhGM-CSF and rhIL-4 were obtained from Biosource (Camarillo, CA) and injected at concentrations of 262.5 U/ml and 112.5 U/ml, respectively. R848 and R837/imiquimod were obtained from InvivoGen (San Diego, CA).
Skin explant preparation and culture
Skin explants from human donors were prepared, injected and cultured as previously described[26;42]. Abdominal resections from healthy donors (Bergman Clinics, Bilthoven, The Netherlands) were obtained with informed consent within 24h after plastic surgery. Cytokines or TLR ligands were diluted in serum-free medium (IMDM) and injected i.d. into the skin in a volume of 20 μl/biopsy: rhGM-CSF (262.5 U/ml), rhIL-4 (112.5 U/ml), R848 (5 μg/ml) and R837. The concentration of R837 was determined per skin donor, based on the size of skin where one sachet of Aldara skin cream was applied on. The amount of soluble R837 that was injected intradermally per biopsy was equal to the amount of Imiquimod in the Aldara skin cream that was applied on the surface of one biopsy. These concentrations were based on optimal maturational effects on monocyte-derived DC (MoDCs) cultured in vitro as reported previously[29]. In each separate experiment, 10-24 biopsies were taken per experimental condition. A 6 mm biopsy punch (Microtec) was immediately taken from the injection site. Biopsies were cultured with the epidermal side up floating in 1 well of a 48 well plate containing 1 ml IMDM supplemented with 10% Fetal Calf Serum (FCS, Greiner Bio-One), 50 U/ml penicillin, 50 μg/ml streptomycin, 10 μg/ml gentamycin and 2 mM L-glutamine (Invitrogen Life Technologies). Biopsies were cultured for 2 days at 370C, 5% CO2, after which biopsies were discarded and migrated cells present in the medium and supernatants were harvested. Supernatants from 48h-cultured full-thickness skin explants were pooled per condition and stored at -200C for cytokine analyses.
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Application of Aldara skin cream
Per donor, 1 sachet of Aldara skin cream was applied on the epidermis of a separate piece of skin and rubbed well into the skin. After 20 min of incubation, excessive cream was removed carefully and serum-free medium was injected i.d. and biopsies were taken as described above.
Phenotypical analysis of crawl-out cells
Phenotypical analysis of emigrated cells was performed by flow cytometry. Cells were washed in PBS supplemented with 1 % BSA and 0.02% NaN3 and incubated for 30 min at 40C in the presence of appropriate dilutions of fluorescent-conjugated mAbs to CD1a, CD14, CD70, CD86, HLA-DR (BD, San Jose, CA), HLA-ABC (ImmunoTools, Friesoythe, Germany) or CD83 (Beckman Coulter Immunotech), or corresponding isotype-matched control mAbs (BD, San Jose, CA). Cell surface expression of Langerin was analyzed using a fluorescent-conjugated mAb (clone DCGM4, Beckman Coulter). The cells were subsequently analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR, USA).
Cytokine ELISA
The levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in the biopsy-conditioned supernatants were quantified using standard sandwich ELISA antibody pairs from Life Technologies following manufacturer’s instructions.
Real-Time PCR
Cells were lysed and mRNA was isolated using an mRNA Capture kit (Roche). cDNA was synthesized using the Reverse Transcription System kit (Promega) following manufacturer’s guidelines. Oligonucleotides were designed using the Primer Express 2.0 software (Applied Biosystems) and synthesized by Invitrogen Life Technologies (Invitrogen). Real-Time PCR analysis was performed as previously described using the SYBR Green method in an ABI 7900HT sequence detection system (Applied Biosystems) [43]. GAPDH was used as an endogenous reference gene.
Antigen presentation to human CD8+ T cell clone
A CD8+ T cell clone specific for MART-126-35 was generated and cultured as described previously [44]. A 15 amino acid long MART-121-35 peptide (YTTAEELAGIGILTV) was i.d. injected in the skin at indicated concentrations. Peptides were mixed with GM- CSF/IL-4 or TLR ligands and co-injected. Aldara skin cream was applied to the skin and allowed to absorb for 20 min at RT followed by i.d. injection of the MART-1 peptide dissolved in IMDM. Peptides were produced by solid phase peptide synthesis using Fmoc chemistry on an automated synthesizer (Protein Technologies, Tuscon Arizona). After 2 days, emigrated HLA-A2+ skin cells were harvested and 20.000 cells/well were incubated in a 96-wells round bottom plate. After extensive
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washing, MART-1-specific CD8+ T cells (100.000/well) were added to the wells. After 24 h, supernatants were taken and IFN-γ levels were measured by sandwich ELISA using specific antibody pairs from Biosource.
Antigen presentation to primary CD8+ T cells
A 15 amino acids long MART-121-35 peptide (YTTAEELAGIGILTV) was i.d. injected in the skin at indicated concentrations. This peptide was co-injected with GM-CSF/IL-4 or TLR ligands as indicated. Aldara skin cream was applied to the skin and allowed to absorb for 20 min at RT followed by i.d. injection of the MART-1 peptide dissolved in IMDM. After 2 days, migrated HLA-A2+ skin cells were harvested and seeded at 50.000 cells/well in duplicate in a 48-wells plate. Allogeneic HLA-A2+ CD8+ T cells, isolated from peripheral blood using a MACS untouched CD8+ T cell isolation kit (Miltenyi, Bergisch Gladbach, Germany), were added (500.000/well) to the DCs together with 10 ng/ml IL-7 (PeproTech, Rocky Hill, NJ). Three days later, cells received fresh medium containing 10 ng/ml IL-7 and 20 IU/ml IL-2 (Chiron Therapeutics Emeryville, CA). At day 7, cells were harvested and stained with a PE- labeled MART-1 tetramer and APC-labeled anti-CD8. Additionally, secretion of IFN-γ and TNF-α (both BD Biosciences) was analyzed. To this end, cells were re-stimulated with 10 μg/ml MART-126-35 peptide in the presence of 10 μg/ml Brefeldin A (Sigma- Aldrich) for 5 h, followed by a staining of the intracellularly expressed cytokines IFN-γ and TNF-α.
Statistical analysis
Results were analyzed using an one-way ANOVA followed by Bonferrroni Multiple Comparison test using GraphPad Prism software (GraphPad Software, San Diego, CA). Results were considered to be significantly different when p<0.05.
Conflict of interest
The authors declare no financial or commercial conflict of interest.
Acknowledgements
We would like to thank the personnel of the Bergman clinic in Bilthoven, The Netherlands for the provision of healthy donor skin. We would like to thank Juan J.
Garcia-Vallejo for the design of the primers used in the real-time PCR experiments.
The present work was funded by KWF (VU2009-2598; C.M. Fehres), NanoNextNL program 3D (W.W.J. Unger), Cancer Centre Amsterdam “Miljoenenronde” (A.J. Van Beelen) and Senternovem (SII071030; W.W.J. Unger and M. Ambrosini).
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Figure S1. Intradermal injection does not disturb the basement membrane between dermis and epidermis. Human skin was intradermally injected with IMDM or not injected (control), whereafter a 6 mm biopsy was taken and cultured for 48 hours. After incubation, the biopsies were fixed and 7 μm cryosections were made. Cryosections were stained with Mayer’s haematoxylin and eosin (H&E). Scale bar: 100 μm.
