Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands

receptor ligands

Weterings, J.J.

Citation

Weterings, J. J. (2008, November 27). Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands. Bio-organic Synthesis, Leiden Institute of Chemistry, Faculty of Science, Leiden University. Retrieved from https://hdl.handle.net/1887/13284

Version: Corrected Publisher’s Version

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Chapter 5

Comparison of R-and S-configured glycerol containing triacylglycerollipids in TLR-2 mediated immune response ¹

Introduction

Toll-like receptor ligands^1a have recently gained increased interest as potential components in synthetic vaccine development.² Toll-like receptors (TLRs) are receptors mainly expressed by cells of the innate immune system, such as granulocytes, macrophages and dendritic cells (DCs), and their function is to sense intruding pathogens through the recognition of the so- called pathogen associated molecular patterns (PAMPs).^{3, 4} Although the TLRs all share highly homologous structures, each TLR recognizes distinct molecular patterns. For instance, TLR2 interacts with lipopeptides⁵, TLR3 with virus-derived double stranded RNA^6-8, while TLR9 recognizes CpG-DNA motifs.⁹ Interestingly, whereas most of the TLRs recognize a narrow ligand repertoire, TLR2 is able to distinguish between a broad spectrum of lipopeptide-derived ligands including lipoteichoic acid¹⁰, di-and triacylated lipopeptides¹¹, peptidoglycan from gram-positive bacteria^{12, 5} and yeast zymosan.¹³ This promiscuity in ligand selection of TLR-2 was recently suggested to be linked to the fact that TLR-2 interacts with other receptors such as TLR-1, TLR-6, CD36 or dectin-1.^14-16

In the previous chapter the superior ability of antigenic peptides covalently linked to Pam₃Cys to induce specific CD8⁺ T cell response as compared to a mixture of non-covalently bound Pam₃Cys and antigenic peptide was described. Both enhanced MHC-I restricted presentation of the antigenic peptide and TLR2-mediated cytokine production (leading to DC maturation) were observed. Peptide-TLR-L conjugates^17-22 are widely regarded as promising leads for vaccine development and one of the attractive features of the development of synthetic peptide-TLR-L constructs is the potential to generate well-defined and enantiopure material.

Such control over stereochemistry allows the execution of so-called structure-activity relationship (SAR) studies, bringing the development of vaccine-based therapies within the fold of classical medicinal chemistry. Moreover, it is likely that synthetic peptide-TLR-L conjugates with advantageous immunomodulatory properties can be brought to the clinic more easily when enantiopure in comparison to structural or stereochemical mixtures. In this sense, the triacyllipopeptide-based constructs have perhaps more potential than the CpG constructs also discussed in Chapter 4 (disregarding the specifics and differences that are behind their immunostimulatory properties) in that they are easily available in enantiopure form. The Pam3Cys constructs described in Chapter 4 are in fact mixtures of diastereomers with respect to the chiral carbon of the glycerol moiety. Most literature studies on synthetic Pam3Cys based TLR2 ligands make use of similar diastereomeric mixtures.²³ However, several studies have revealed that enantiopure Pam3Cys derivatives containing R-configured glycerol (that is, the TLR2 ligands alone without antigenic peptide covalently bound) are more potent inducers of cytokine (IL6, IL8) production and are better capable of stimulating antibody production in mice when injected in combination with antigenic proteins.^24-26 This raises the question whether enantiopure Pam₃Cys-peptide conjugates containing R-configured glycerol are more potent TLR2-agonists, give enhanced MHC I-restricted antigen presentation, or both. In this chapter the synthesis and evaluation of the set of enantiopure

Pam₃Cys-peptide conjugates (3, 4) having either the 2R or 2S configuration is described.

Results

O C15H31

O O C15H31

O S

N H

O 2

C15H31

O

H

N SKKK KDEVSG LEQLES IINFEK L

3

O C15H31

O O C15H31

O S

FmocHN O 2

OH

1 1

1

O C15H31

O O C15H31

O S

FmocHN O 2

OH 1

2

O C15H31

O O C15H31

O S

N H

O 2

C15H31

O

H

N SKKK KDEVSG LEQLES IINFEK L

4 1

Figure 5.1: Structure of Pam3Cys peptide conjugates

The diastereomerically pure conjugates Pam(R)-OVA_247-264 3 and Pam(S)-OVA_247-264 4 (Figure 5.1) were prepared on solid phase starting from known²⁷ chirally pure Fmoc-building blocks 1 and 2 (Figure 5.1) according to Scheme 5.1. The immobilized, side-chain protected peptide fragment 6 was obtained by automated Fmoc-based solid phase peptide synthesis.²⁸ After Fmoc deprotection using 20% piperidine in NMP coupling of either the R or S (1 or 2 respectively) compounds onto the resin bound peptide 6 was accomplished using PyBOP and DiPEA. The resin bound peptide R-FmocPam₂CysSK₄-OVA_247-264 7 was Fmoc deprotected and palmitoyl carboxylic acid chloride was coupled onto the N-terminus aided by pyridine giving 9. Release from the resin was achieved by acid treatment (TFA/TIS/H2O, 95/2.5/2.5), after which the crude peptide was precipitated in diethylether. The crude was dried by N2 flow and purified by RP-HPLC. It was found that HPLC purification of the final lipopeptides was only feasible when the relatively polar CN-stationary phase was applied and that the standard C18 and C4 stationary phases gave prohibitively long retention times. Pam(R)-OVA247-264 3 and Pam(S)-OVA247-264 4 were obtained in yields of 44% and 40% respectively, based on the theoretical loading of resin 6. Construct 4 was obtained via a similar procedure as used for 3 utilizing S-FmocPam2Cys-OH 2.

O C15H31

O O C15H31

S O

FmocHN O H

O 2

23

O C15H31

O O C15H31

S O

FmocHN O 2

HN S KKKK DE VSGLE QLESIINFEKL

8 O

O

1.TFA/TiS/H2O 2. RP-HPLC 1

1 Fmoc

HN O O

S KKKK DEV SGLEQ LESIINFEKL HN

Fmoc

O

O 6

Fmo c SPPS Fmo c-Le u-S-PHB resin

20% pipe ridine in NMP

O C15H31

O O C15H31

S O

FmocHN O H

O 2

23 1

5

1 2

PyB oP, DiPEA PyBoP, DiPE A

O C15H31

O O C15H31

S O

FmocHN O 2

HN S KKKK DE VSGLE QLESIINFEKL

7 O

O 1

1.TFA/TiS/H2O 2. RP-HPLC

O C15H31

O O C15H31

S O

N

H O

2

C15H31 O

HN S KKKK DE VSGLE QLESIINFEKL

10 O

O 1

O C15H31

O O C15H31

S O

N

H O

2

C15H31 O

HN S KKKK DE VSGLE QLESIINFEKL

9 O

O 1

1. 20% pip eridine in NMP 2. Pa m-Cl/pyridine 1. 20% pip eridine in NMP

2. Pa m-Cl/pyridine

O C15H31

O O C15H31

S O

N

H O

2

C15H31 O

HN S KKKK DE VSGLE QLESIINFEKL

3 O H

O 1

O C15H31

O O C15H31

S O

N

H O

2

C15H31 O

HN S KKKK DE VSGLE QLESIINFEKL

4 O H

O 1

44% overall 40% overall

Scheme 5.1: Synthesis of stereochemically pure Pam3Cys peptide conjugates 3 and 4

The effect of the stereochemistry at C(2) on dendritic cell maturation.

Upregulation of the surface markers CD40, CD86 and cytokine IL-12 release by DCs were determined by the following procedure. Compounds Pam(R)-OVA_247-264 3, Pam(S)-OVA_247-

264 4 and the epimeric mixture Pam(RS)-OVA247-264 3/4 prepared by mixing compound 3 and compound 4 (Figure 5.1) were applied in vitro and in vivo in PBS. DCs were stimulated for 48h. with either the Pam(R)-OVA_247-264 3, the Pam(S)-OVA247-264 conjugate 4, or a 50/50

mixture of Pam(RS)-OVA_247-264 conjugate 3/4 and the upregulation of surface markers associated with dendritic cell maturation was analysed by flow cytometry.

Figure 5.2: Activation of dendritic cells

Dendritic cells were stimulated for 48h. with either the Pam(R)-OVA_247-264 3 (black bars) Pam(S)-OVA_247-264 4 (white bars), the mixture of R and S conjugates 3/4 (grey bars) or with LPS (10ug/ml hatch bars). The indicated concentration (1 μM, 0.5 μM, or 0.25 μM) on the x- axis refers to the final concentration of the individual TLR-2L stereomers, thus, a 1 μM of the RS mixture contain 1 μM of Pam(R)-OVA247-264 and 1 μM of Pam(S)-OVA247-264, whereas the final concentration of OVA247-264 peptide is 2 μM. Dendritic cells were stained with mAbs specific for the cell surface markers as indicated (A) CD40, (B) CD86 and subjected to flow cytometry. Shown is the mean fluorescence intensity (MFI) of the individual proteins gated on live cells. (C) Histogram overlay, showing CD40 expression at 1 μM final concentration;

top panel: untreated control (neg, filled histogram), the Pam(RS)-OVA247-264 3/4 (RS-conj., solid line); bottom panel: Pam(S)-OVA247-264 4, the Pam(R)-OVA247-264 3. Data are representative of two independent experiments. (D) and (E) supernatant from dendritic cells incubated for 24 h with the indicated compounds were subjected to IL12 ELISA as outlined in the material and method section. The concentration displayed on the x-axis in panel D refers to the molar peptide concentration. In panel E the x-axis displays the molar concentration of the Pam(R) stereomer 3.

As shown in Figure 5.2, treatment with the Pam(R)-OVA247-264 3 led to a 4-5 fold higher surface expression of CD40 and a 2-3 fold higher surface expression of CD86 compared to cells stimulated with the Pam(S)-OVA_247-264 conjugate 4. Stimulation of dendritic cells with the mixture of the epimers (Pam(RS)-OVA_247-264) 3/4 resulted in similar maturation profile as that observed with the Pam(R)-OVA_247-264 3 alone. Furthermore, IL12 production was significantly lower in dendritic cells matured by the Pam(S)-OVA_247-264 4 compared to the Pam(R)-OVA_247-264 3(Figure 5.3D). On the other hand comparing the potency of the Pam(R)- OVA_247-264 3 with that of the epimeric mixture 3/4 (containing 1:1 ratio of Pam(R) and Pam(S)) no significant difference was observed in the ability to produce IL12 (Figure 5.3E).

The effect of the stereochemistry of the constructs on antigen presentation in vitro. Next, the capacity of the Pam(R)-OVA_247-264 3and the Pam(S)-OVA_247-264 4 conjugate to support MHC class I antigen presentation in vitro was examined.

Figure 5.3: MHC class I antigen presentation

(A) Dendritic cells loaded either with Pam(R)-OVA247-264 3 (black bars), Pam(S)-OVA247-264 4 (grey bars), or the racemic mixture Pam(RS)-OVA247-264 3/4 (white bars). (B) DCs were left untreated (grey bars), or pre-treated for 60 min with 50 μM MDC before addition of 0.5 μM of either Pam(R)-OVA_247-264 3 or Pam(S)-OVA_247-264 4 or the minimal CTL epitope SIINFEKL (OVA8).

As can be seen in Figure 5.3 A similar levels of antigen presentation were observed for the two epimeric forms of the conjugates. These results imply that the Pam(R)-OVA_247-264 3and the Pam(S)-OVA_247-264 4conjugates are equally well taken up and processed by the dendritic cells. Therefore the uptake route of the two independent stereomers was investigated. To this end dendritic cells were pre-treated with monodansylcadaverin, a specific inhibitor of clathrin-dependent endocytosis^{29, 30} before incubation with the peptide conjugates. Antigen presentation of both epimer conjugates was inhibited to the same extent (50-60% reduction), Taken together, these results show that although the chirality at the C2 position is important for interacting with TLR-2 and thereby activating the DCs, it does not influence the uptake efficiency and mechanism.

The effect of the stereochemistry of the constructs on T cell induction in vivo.

Since the results from the in vitro antigen presentation studies revealed that there was no significant difference between the Pam(R)-OVA_247-264 3and Pam(S)-OVA_247-2644 conjugate in consecutive antigen presentation on MHC class I, the implication of the R and S configuration at C-2 of the glycerol moiety on their ability to induce an endogenous specific CD8⁺ T-cell- response was investigated next. Naïve C57BL/6 mice were injected subcutaneous either with the R- conjugate 3, the S-conjugate 4, the RS-mixture 3/4 or free peptide SIINFEKL. After 12 days, induction of OVA_257-264-specific CD8⁺ T-cells was analyzed by quantification of in vitro stimulated T cells with H-2K^b-peptide tetramers. A significantly stronger induction of OVA257-264-specific T-cells was observed in mice injected with the Pam(R)-OVA247-264 3 compared to mice injected with the Pam(S)-OVA247-264 conjugate 4 (Figure 5.4 A). The percentage of specific T-cells induced by the S-epimer was in the same order as in mice injected with the non-conjugated OVA247-264 peptide. Finally, the functionality of the induced T-cells by their ability to produce IFNγ by intracellular cytokine staining was investigated. As can be seen from Figure 5.4 B a more than 4-fold increase in IFN-γ production was observed in T-cells from mice injected with the Pam(R)-OVA247-264 compared to mice injected with the Pam(S)-OVA247-264 4 further confirming that only T-cells induced by the Pam(R)-OVA247-264

conjugate 3 are functional.

Figure 5.4: Priming of endogenous CD8+ T-cells

(A). Naïve C57/B6 mice were injected with either 2.5 nmol Pam(R)-OVA_247-264 3, 2.5 nmol Pam(S)-OVA_247-264 4_, or a mixture of Pam(R)-OVA_247-264 3and Pam(S)-OVA_247-264 4_, (each 1.25nmol), 2.5nmol OVA_247-264 alone. (B). IFN-production in specific T-cells

These results show that the chirality at the C2 position is of importance for facilitating maturation of dendritic cells which directly relates to the superior ability of DCs to induce antigen-specific CD8⁺ T-cells.

Discussion

Chapter 4 revealed that synthetic antigenic peptides covalently linked to the TLR2-ligand Pam₃CysSK₄, present as a mixture of epimers at C-2 of the glycerol moiety of Pam₃Cys, were efficiently targeted into the MHC class I cross-presentation pathway¹⁹ and were able to strongly enhance the induction of specific CD8⁺ T-cells. In this chapter, the effect of configuration at the C-2 position of the glycerol moiety of the Pam₃CysSK₄ linked to OVA_247-264 antigenic peptide on maturation and activation of dendritic cells as well as the ability to induce specific CD8⁺ T-cells was investigated. It was found that Pam(R)-OVA_247-264 3 was superior in facilitating activation and maturation of dendritic cells (Figure 5.2) as well as inducing specific CD8⁺ T-cells (Figure 5.4). These findings are in line with previous reports showing that free Pam3Cys lipopeptides of the R-configuration at the glycerol position are more biologically active than those with S-configuration.^{23, 26, 31} The new insights described in this chapter help to explain the immunological mechanisms underlying the described properties. No significant differences between the Pam(R)-OVA247-264 3 and the Pam(S)-OVA247-264 conjugate 4 in terms of MHC class I antigen presentation in vitro (Figure 5.3) were observed, suggesting that both (conjugate) steroisomers are equally well internalized and processed by the dendritic cells. Although at first sight these findings seem to contradict each other the results can be explained by the fact that the B3Z cell assay is independent of co-stimulation.³² The current observation with the S-epimer conjugate which showed similar level of antigen presentation as the R-epimer, but the impaired DC activation and poor induction of specific CD8⁺ T cells by the S-epimer is in line with the study detailed in the former chapter comparing the immunological activity of the highly potent CpG peptide conjugate to the non-functional GpC peptide conjugate.¹⁹ This further strengthens the proposed mechanism that joined entry of antigen and DC maturation is a prerequisite for an optimal induction of specific CD8⁺ T cell response. Moreover, it was shown before that the uptake of the diastereomeric mixture of Pam(RS)-OVA_247-264 conjugates occurs in a receptor mediated manner though independent of the surface expressed TLR2.¹⁹ The antigen presentation data suggest that the internalisation efficiency of the R and S-conjugates are similar thus this would imply that the diastereomeric composition does not appear to be important for binding to the (to date unidentified) receptor involved in the internalisation of the R- and S-conjugates. DC maturation studies by either the R or S conjugate however, show that the glycerol moiety of the former is responsible for higher cytokine levels, CD40 and CD86 upregulation compared to the S epimer, One explanation for this occurrence would be that the R configuration of the glycerol moiety in Pam3Cys results in a better binding or

Experimental Section

General methods: General methods: All chemicals and solvents used in the solid phase peptide synthesis, except of HCTU, pam₃Cys-OH, palmitoyl carboxylic acid chloride and PYBOP were from Biosolve (The Netherlands) and used as received. HCTU was purchased from IRIS Biotech GmbH (Germany) and Pam₃Cys-OH bought at Bachem Distribution GmbH (Germany). PYBOP was purchased at Multisynthech GmbH. Palmitoyl carboxylic acid chloride was received from Alldrich. Fmoc-amino acids were from SENN Chemicals or from MultiSynTech GmbH (Germany). Tentagel based resins were bought at Rapp Polymere GmbH (Germany). Mass spectra were recorded on a PE/SCIEX API 165 (Perkin-Elmer).

Analytical LC/MS of non-lipidated peptides was conducted on a JASCO system using an Alltima C18 analytical column (5μ particle size, flow: 1.0 ml/min). Analytical LC/MS of Pam3Cys-peptides was conducted on a JASCO system using an Alltima CN analytical column (5μ particle size, flow: 1.0 ml/min). Absorbance was measured at 214 and 254 nm. Solvent system: A: 100% water, B: 100% Acetonitrile, C: 1% TFA. Gradients of B in 10% C were applied over 13.5 or 26 minutes unless stated otherwise. Purifications of Pam3Cys conjugates were conducted on a BioCAD “Vision” automated HPLC system (PerSeptive Biosystems, inc.), supplied with a semipreparative Phenomenex® CN Luna column (5μ, 250mm x 100 mm, flow 4 ml/min.). Solvent system: A: 100% water, B: 100% acetonitrile, C: 1% TFA.

Gradients of B in A/C were applied over 3 CV. Concentration of C was maintained at 10%

throughout. The UV absorption was measured with a Varian DMS 200 UV visible spectrophotometer. MALDI-TOF spectra were recorded on a Voyager-DE PRO mass spectrometer (Perseptive Biosystems, inc.).

The solid-phase peptide synthesis was performed on a CS Bio 336 automated instrument (CS Bio, California, USA) applying Fmoc based protocol starting from preloaded Fmoc-Leu- PHB-Tentagel resin. The synthesis was performed on a 50 or 250 μmol scale according to established methods²⁸. The consecutive steps performed in each cycle were:

1) Deprotection of the Fmoc-group with 20% piperidine in NMP for 15 min; 2) DMF wash;

3) Coupling of the appropriate amino acid applying a five-fold excess. Generally, the Fmoc amino acid (0.25 mmol) was dissolved in 0.25 M HCTU in NMP (1 ml), the resulting solution was transferred to the reaction vessel followed by 0.5 ml of 1 M DIPEA in NMP to initiate the coupling. The reaction vessel was then shaken for 45 min. 4) DMF wash; 5) Capping with 0.5 M NMP solution of acetic anhydride in presence of 0.5 mmol DIPEA 6) DMF wash.The Fmoc amino acids applied in the syntheses were: Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,

FmocAsp(Ot-Bu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Ile- OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Ser-OH and Fmoc-Val-OH

Fmoc-Ser(O^tBu)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Asp(O^tBu)-Glu(O^tBu)-Val- Ser(^tBu)-Gly-Leu-Glu(O^tBu)-Gln(Trt)-Leu-Glu(O^tBu)-Ser(^tBu)-Ile-Ile-Asn(Trt)-Phe- Glu(O^tBu)-Lys(Boc)-Leu-Tentagel-S-PHB (6)

Resin bound peptide 6 was prepared using a CS Bio 336 automated instrument applying fmoc based protocol starting from 50 μmol of Tentagel S PHB resin (200 mg, loading 0.25 mmol/g). A small aliquot of the resin was transferred to a glass tube and treated for 2 hours with a cleavage cocktail TFA/TiS/H2O (95/5/5). The solution was filtered into cold diethylether and the resin was washed with neat TFA. The diethylether was centrifuged, removed and the precipitate was washed with diethylether. After centrifugation, the ether was removed and the precipitate was air dried and dissolved in ^tBuOH/ACN/H2O (3/1/1, v/v).

LCMS: 10-90% B, Rt = 4.55 min; ESI-MS: [M+H]⁺: 2664.0 (calculated 2664.0), [M+H]²⁺: 1332.4 (calculated 1332.5), [M+H]³⁺: 888.6 (calculated 888.7).

Synthesis of N-Palmitoyl-S-[2,3-Bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine-Ser-Lys-Lys- Lys-Lys-Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-OH (1) (Pam(R)-OVA_247-264) 3

To a mixture of resin bound peptide 6 (see Scheme 5.1), (100 mg resin, 0.015 mmol, theoretical loading 0.15 mmol/g) in 500 μl DCM/NMP 1/1 was added (R,R)-FmocPam2Cys- OH 1 (2 eq., 26,7 mg, 30 μmol), PyBoP (2 eq., 16 mg, 30 μmol) and DiPEA (1,1 eq., 2.8 μL, followed by 0,9 eq., 2,3 μL after 30 min). After shaking for 4 hours, the resin was washed with NMP and DCM, followed by drying (flow N₂). Remaining free amines were capped with 0.5 M Ac₂O / 0.125 M DiPEA solution in NMP (1 mL, 5 min). Fmoc was cleaved (20%

piperidine in NMP,1 mL, 20 min) and the resulting amine was palmitoylated by addition of palmitoyl chloride (10 eq., 46 μL) to the resin bound peptide in NMP/pyridine 1/1 (1 mL) followed by shaking for 2.5 h. The resin was washed with NMP, DCM and dried by N2 flow.

The resulting conjugate was cleaved from the resin with TFA/TiS/H2O, 95/2.5/2.5 (4 ml per 100 mg resin, 2 h), the resin was filtered off and washed with neat TFA. The resulting filtrates were collected in cold freshly distilled Et2O, the ether layer was centrifuged for 5 min. at 4000 rpm and decanted. The precipitate was dried by an N2 flow and dissolved in AcOH/H2O/ACN 1/1/1. (6 mL). The crude peptide was subjected to CN-HPLC on a Phenomenex Luna semipreparative column running a gradient of 45%-70% B in A/C. Pure peptide fractions

B, Rt = 7.18 min; ESI-MS: [M+H]²⁺: 1778.4 (calculated 1778.9), [M+H]³⁺: 1186.2 (calculated 1186.3). MALDI-TOF: [M+H]⁺:3561.4 (calculated 3556.9).

Synthesis of N-Palmitoyl-S-[2,3-Bis(palmitoyloxy)-(2S)-propyl]-(R)-cysteine-Asp-Glu- Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-OH (4) (Pam(S)-

OVA247-264) Prepared and purified according to the same procedure as used for 3 (Scheme

5.1). Yield: 21.5 mg (6.0 μmol, 40%). LCMS: 10-90% B, Rt = 7.10 min; ESI-MS: [M+H]²⁺: 1778.4 (calcld. 1778.9), [M+H]³⁺: 1186.2 (calculated 1186.3). MALDI-TOF: [M+H]⁺:3564.8 (calculated 3556.9).

Cell lines, mouse strain and chemicals D1 cell line, a long term growth factor-dependent immature splenic DCs line derived from B6 (H-2^b) mice, was cultured as described elsewhere.³² B3Z is a T-cell hydridoma specific for the H-2K^b CTL epitope SIINFEKL, which expresses a β-galactosidase construct under the regulation of the NF-AT element from the IL2 promoter.³² EG7³³ (EL4-OVA) was cultured in complete medium with 400 μg/mL G418 (Gibco). C57BL/6 (B6; H-2^b) mice were obtained from Charles River Laboratories. All mice were bred in the specific pathogen-free animal facility of the Leiden University Medical Center. Chlorophenol red-ß-D-galactopyranoside (CPRG) was purchased from Calbiochem.

Monodansylcadaverin (MDC) was obtained from Sigma-Aldrich.

IL-12p40 ELISA. Dendritic cells (4x10⁴) were plated into 96-well round bottom plate, and incubated for 24h. with the compounds indicated in the figure legends. To generate 1 μM diastereomic mixture 3/4 composed of Pam(R)-conjugate and Pam(S)-conjugate, 1 μM Pam(R)-conjugate 3 was mixed with 1 μM Pam(S)-conjugate 4. Supernatants were collected, and tested for IL12 p40/p70 content using a standard sandwich ELISA, as previous described.³²

Flow cytometry For analysing the effect of the different compounds on dendritic cell phenotypic profile, dendritic cells were incubated with the compounds at a final concentration of 0,25 μM, 0.5 μM or 1 μM for 48 h. Subsequently, cells were harvested and resuspended in FACS buffer (PBS / 0.1% BSA), followed by incubating for 20 min with the following panel of monoclonal antibodies FITC-K^b (M5/114.5), FITC-anti-CD86 (clone GL-1), PE-anti-CD40 (clone 3/23), APC-I-A^b (clone 1C10). Cells were washed twice before propidium iodide was added and samples were subjected to flow cytometry measurements.

MHC-class I-restricted antigen presentation assay DCs were incubated for 5 h, with either the Pam(R)-OVA_247-264 3 configuration conjugate, the Pam(S)-OVA_247-264 4 configuration conjugate, or a mixture of the Pam(R)- and Pam(S)-conjugate at the concentrations indicated in the figure legends. In some experiments cells were treated for 60 min with 50 μM MDC prior to incubation with the peptides. Cells were then washed five times with medium before the T-cell hybridoma B3Z cells were added and incubated for 16h. at 37^oC.

Antigen presentation of the ovalbumin cytotoxic T-cell epitope, SIINFEKL (OVA257-264) in H-2K^b was detected by activation of B3Z cells measured by a colorimetric assay using chlorophenol red- -D-galactopyranoside as substrate to detect lacZ activity in B3Z lysates, as described.³²

Priming of endogenous naïve CD8+ T-cells To determine the endogenous CTL response, 2.5 nmol of the different compounds was injected s.c. into naïve C57BL/6 mice in a final volume of 200 μl PBS. After 10 days mice were sacrificed and spleen cells were stimulated in vitro by plating 5x10⁶ splenocytes with 0.5x10⁶ Mytomycin C (Kyowa) treated (50 μg/ml, 1 hour at 37 degrees) and irradiated (4000 rad) EG7 cell line (EL4-OVA), in the absence of additional cytokines. After seven days viable splenocytes were isolated over a ficoll gradient and stained for H-2K^b Tetramer (TM)-OVA_257-264 and CD8b2 (clone 53-5.8), and propidium iodide to exclude dead cells.

Intracellular cytokine staining Production of IFNγ was measured exactly as previously described¹⁹ Briefly, an aliquot of spleen cells after re-stimulation and ficoll purification (see above) were subjected to stimulation in vitro with or without 1 μg/ml OVA257-264 peptide (H2- K^b restricted SIINFEKL) overnight in the presence of GolgiPlug (BD Pharmingen, San Diego, CA, USA). Cells were then washed twice with FACS buffer and stained with PE- conjugated monoclonal rat anti-mouse CD8b2 antibody. Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Pharmingen, San Diego, CA, USA). Intracellular IFN-γ was stained with APC- conjugated rat anti-mouse IFN-γ. All antibodies were purchased from BD Pharmingen. Flow cytometry analysis was performed using FACSCalibur with CELLQuest software (BD Biosciences, Mountain View, CA, USA). Splenocytes without peptide stimulation were used as a negative control.

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5. A. Yoshimura, E. Lien,E, R.R. Ingalls, E. Tuomanen, R. Dziarski, D. Golenbock, J. Immunol., 1999, 163, 1-5.

6. A.J. O'Neill and A.G. Bowie, Nat. Rev. Immunol., 2007, 7, 353-364.

7. S.S. Diebold, T. Kaisho, H. Hemmi, S. Akira, C. Reis e Sousa, Science, 2004, 303, 1529-1531.

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