Previously, we have shown that conjugation of a palmitic chain via a thioester bond to a cysteine residue in weakly or nonencephalitogenic or neuritogenic peptides markedly enhances their ability to induce autoimmune disease in an MHC class II–restricted manner. From those studies, however, it was not clear whether thiopalmitoylation of the peptides was merely enhancing their disease-inducing potential or whether the lipid was itself playing a pathogenic role. To investigate this further, we have now tested the effects of thiopalmitoylation on MHC class II–restricted altered peptide ligands (APLs), which are normally protective in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. We hypothesized that if thiopalmitoylation of a peptide merely enhances its innate potential, then thiopalmitoylated APLs (S-palmAPLs) should show enhanced protective effects. Alternatively, if thiopalmitoylation itself can make a peptide pathogenic, then S-palmAPLs should have decreased therapeutic potential. We synthesized APLs and corresponding S-palmAPLs and showed that the S-palmAPLs were much more effective than the nonconjugated APL at inhibiting the development of experimental autoimmune encephalomyelitis. This was due to several features of the S-palmAPL:S-palmAPL–primed cells show an enhanced ability to proliferate and produce the anti-inflammatory cytokine, IL-10, in vitro. Furthermore, the bioavailability of S-palmAPL was greatly enhanced, compared with the nonpalmitoylated APL, and S-palm APL was taken up more rapidly into dendritic cells and channeled into the MHC class II processing pathway. These results show that thiopalmitoylation of MHC class II–restricted peptides is a simple way to enhance their effects in vivo and could have wide therapeutic application.

The role that lipid posttranslational modifications to proteins play in the immune response to those proteins is still poorly understood. We have been interested in the role that one such modification, thiopalmitoylation (attachment of palmitic acid via a thioester bond to cysteine residues of a protein), might play in immune responses directed against myelin proteolipid protein (PLP), a major component of CNS myelin and a putative autoantigen in the demyelinating disease, multiple sclerosis (MS). PLP is normally thiopalmitoylated at up to six sites at a ratio of ∼3 mol of lipid per mole of protein; however, it is known that the degree of thiopalmitoylation of PLP increases markedly during the process of demyelination (1), and thus, during myelin breakdown in MS, there is the potential for thiopalmitoylated PLP peptides to be released. Previously, we have shown that thiopalmitoylation of specific PLP peptides markedly enhances their encephalitogenicity in an experimental autoimmune encephalomyelitis (EAE) model of MS (2). Similarly, thiopalmitoylation of peptides from P0, a major protein of peripheral nervous system myelin, enhances their neuritogenic potential in experimental autoimmune neuritis (3). The enhanced pathogenicity of the thiopalmitoylated peptides appears to be because of their increased uptake (compared with nonpalmitoylated peptide) into the MHC class II presentation pathway (4, 5). One question arising from the above studies is whether the thiopalmitoylation is merely enhancing the innate potential of these peptides to be pathogenic or whether thiopalmitoylation of peptides that have little or no disease-inducing potential can actually make them pathogenic. To address this question, we decided to test the effects of thiopalmitoylation on MHC class II–restricted altered peptide ligands (APLs) that are normally protective in EAE. We hypothesized that if thiopalmitoylation of a peptide merely enhances its innate potential, then thiopalmitoylated APLs (S-palmAPLs) should show enhanced therapeutic efficacy. Conversely, if thiopalmitoylation itself can make a peptide pathogenic, then S-palmAPLs should have decreased therapeutic potential and/or should become pathogenic.

APLs are analogs of immunogenic peptides that have been modified at one or more key TCR contact positions to inhibit or modulate the response of T cells that recognize the cognate ligand from which the APLs are derived (6, 7). Depending on their particular sequence, APLs can 1) induce responses in T cells similar to those induced by the cognate ligand, 2) induce some, but not all, of the signals and effector functions downstream of the TCR that are generated by the cognate ligand, leading to altered patterns of cytokine production or to anergy, or 3) antagonize T cell activation by generating a dominant negative signal (6, 813). APLs with antagonistic and immunomodulatory properties have the potential to therapeutically suppress pathogenic T cell–mediated autoimmune responses.

In experimental animals, APLs have been found to effectively and reliably dampen autoimmune responses, particularly when used at high concentrations (8, 9, 14). Several well-defined APLs have been derived from the immunodominant epitopes of PLP for SJL mice, PLP139–151 and PLP178–191 (15, 16). For PLP139–151, two APLs have been studied in detail, namely Q144, which has a substitution of glutamine (Q) for tryptophan at position 144 (9), and the double-substituted APL, L144R147 (leucine at position 144 and arginine at position 147) (8, 9). The APL related to PLP178–191 is known as A188 and has an alanine (A) at residue 188 instead of phenylalanine (14). Both Q144 and A188 appear to exert their in vivo effects predominantly through increasing levels of the cytokines IL-4, IL-10, and IFN-γ (9, 14).

In the current study, we compared the effects of nonpalmitoylated APLs (A188 and Q144) and the corresponding S-palmAPLs to determine whether S-palmAPL are themselves encephalitogenic and lose the therapeutic efficacy of the nonpalmitoylated APLs, or whether the protective effects of the APL are enhanced by thiopalmitoylation. We show that S-palmAPLs show greatly enhanced protective effects, and that this appears to occur because of the effects of thiopalmitoylation on stability of the peptide in serum, increased uptake of the APL into the MHC class II presentation pathway, and induction of greatly enhanced levels of IL-10 following S-palmAPL administration. The enhanced levels of IL-10 also appear to induce bystander effects, enabling S-palmAPL to inhibit the encephalitogenicity of unrelated peptides in vivo.

Peptides (Table I) were manually synthesized by solid phase-synthesis using the Fmoc/tBu strategy. Thiopalmitoylation of residue Cys at position 183 was performed on the resin-bound peptide after selective deprotection of the Cys(Mmt) side chain (17). The biotin-labeled thiopalmitoylated peptides were synthesized as we have described previously (4). After cleavage from the resin, the crude peptides were lyophilized and purified by reversed phase HPLC. The purity of the peptides was assessed by analytical HPLC, and their identities were confirmed by MALDI-TOF mass spectrometry.

Table I.
Designation and sequences of the synthetic peptides used in this study
Peptide DesignationSequence
PLP178–191 (native sequence) NTWTTCQSIAFPSK 
A188 NTWTTCQSIAAPSK 
S-palmA188 NTWTTC(Palm)QSIAAPSK 
A188-biotin NTWTTCQSIAAPSK(Biot) 
S-palmA188-biotin NTWTTC(Palm)QSIAAPSK(Biot) 
PLP139–151 (native sequence) HCLGKWLGHPDKF 
Q144 HCLGKQLGHPDFK 
S-palmQ144 HC(Palm)GKQLGHPDFK 
Peptide DesignationSequence
PLP178–191 (native sequence) NTWTTCQSIAFPSK 
A188 NTWTTCQSIAAPSK 
S-palmA188 NTWTTC(Palm)QSIAAPSK 
A188-biotin NTWTTCQSIAAPSK(Biot) 
S-palmA188-biotin NTWTTC(Palm)QSIAAPSK(Biot) 
PLP139–151 (native sequence) HCLGKWLGHPDKF 
Q144 HCLGKQLGHPDFK 
S-palmQ144 HC(Palm)GKQLGHPDFK 

For injections into mice and for in vitro tissue culture, stock solutions of 5 mg/ml peptides dissolved in 0.2 M acetic acid were further diluted in PBS or tissue culture medium immediately prior to use. For the uptake experiments, stock solutions of pure biotinylated peptides were made up at a concentration of 5 mg/ml in H2O (A188-Biot) or in 30% DMSO (S-palmA188-Biot).

SJL/J mice were purchased from the Animal Resources Centre (Murdoch, WA, Australia) and were immunized at 6–8 wk of age. Mice were maintained and used in accordance with the University of Queensland ethical guidelines for use of experimental animals, and the experimental protocol was approved by the Animal Ethics Committee of the University of Queensland.

For induction of EAE, 32–65 μmol peptide and 400 μg Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in an emulsion consisting of equal volumes of PBS and CFA was injected s.c. on the back of the mouse. Each mouse also received 300 ng Bordetella pertussis toxin (Sapphire Biosciences, Redfern, NSW, Australia) i.v. on days 0 and 3. Clinical assessment (weighing and observation) was conducted daily from day 7 postinjection. Mice were scored according to the following criteria: 0, no disease; 1, decreased tail tone or slightly clumsy gait; 2, tail atony and/or moderately clumsy gait and/or poor righting ability; 3, hind limb weakness; 4, hind limb paralysis; and 5, moribund state. For coimmunization experiments, APLs or S-palmAPLs, at different molar ratios, were included in the emulsion with the adjuvant.

Mice were immunized s.c. with 32 μmol PLP178–191, A188, or S-palmA188 peptide in an emulsion consisting of equal volumes of PBS and CFA (Difco). Ten days after immunization, mice were euthanized, and axillary and inguinal lymph nodes were removed and teased apart. Lymph node cells (LNC) pooled from at least two mice were washed twice with sterile PBS and stained with 2 μM CFSE (Invitrogen Life Technologies, Melbourne, VIC, Australia) for 30 min at 37°C. Cells were cultured at 4 × 106/ml in phenol red-free RPMI 1640 medium (Life Technologies) containing 10% Serum Supreme (Lonza Australia, Melbourne, VIC, Australia), 2 mM HEPES (Lonza), 50 μM 2-ME (Sigma-Aldrich, Sydney, NSW, Australia), and 2 mM l-glutamine (Lonza) in the presence or absence of Ag (25 μg/ml) for 5 d. Wells with no Ag and Con A (2 μg/ml) were used as negative and positive controls, respectively.

After 5 d, cells were harvested and washed with PBS containing 1% FCS and 0.01% sodium azide (wash buffer) (Sigma-Aldrich), before staining with PerCP-labeled anti-CD4 or anti-CD8 Abs (BD Biosciences, Sydney, Australia) for 1 h at 4°C in the dark. PerCP-labeled isotype-matched primary Abs (BD Biosciences) were used as controls. Stained cells were analyzed on a FACSCalibur flow cytometer using CellQuest Software (BD Biosciences). All staining profiles were based on lymphocyte-gated cells, as determined by forward and side scatter properties. The results are expressed as a percentage of CD4+ or CD8+ cells in the sample that are dividing (reactive) in response to the Ag, and a cell division index was calculated by dividing the percentage of dividing cells in the Ag-stimulated group by the percentage of dividing cells in the control group (no Ag). Assays were done in triplicate.

Culture supernatants were collected after 72 h from LNC cultured with Ag as for proliferation assays, and stored frozen at −80°C until use. Ready-Set-Go sandwich ELISA kits (eBioscience, San Diego, CA) specific for IL-10, IL-17, and IFN-γ were used to determine the concentration of cytokines in the supernatants, as per the manufacturers’ instructions. Each supernatant was tested in triplicate, and the concentrations of cytokines were determined from comparison against standard curves prepared from serial dilutions of recombinant cytokines tested on the same plate as the test samples. Results from duplicate assays were calculated and graphed as a fold change in concentration of stimulated cells compared with unstimulated/background (no Ag) levels.

Levels of the cytokines IL-10 and IFN-γ in culture supernatants from control or Ag-stimulated LNC of mice coimmunized with PLP139–151 and A188 or S-palmA188 were quantitated using Cytokine Bead Array (CBA) Mouse IL-10 (bead C4) and IFN-γ (bead A4) sets (BD Biosciences, Franklin Lakes, NJ), as per the manufacturer’s instructions. Briefly, undiluted culture supernatant was incubated with cytokine capture Ab–bead complexes and PE-conjugated Ab (specific for beads) for 2 h. Data were acquired using a BD FACSCalibur flow cytometer and analyzed using FCAP Array software v3.0.1 (BD Biosciences). The concentrations of each cytokine (picograms per milliliter) were determined from a standard curve generated using 10 serial dilutions of cytokine standards.

Peptide (500 μg) was dissolved in 900 μl 0.05 M phosphate buffer (pH 7.2) containing 1% acetonitrile. FCS (10% serum, v/v) was added (100 μl). The reaction mixture was incubated at 37°C. At different incubation times, a 100-μl aliquot was collected, and protease activities were blocked by adding 2 vol of acetonitrile (200 μl). After centrifugation at 3000 rpm for 5 min, the supernatant was analyzed by reversed phase HPLC. In these conditions, no peptide precipitated. A control sample was run in the absence of peptide.

Uptake and presentation of APL and S-palmAPL by dendritic cells.

Dendritic cells (DC) were isolated from spleens of SJL/J mice (Janvier, Le Genest-Saint-Isle, France) using positive selection with CD11c+ MicroBeads (N418) (Miltenyi Biotec, Paris, France), as per the manufacturer’s instructions. DC were resuspended in RPMI 1640 medium containing 5% FCS and 5 ng/ml GM-CSF and allowed to adhere to glass coverslips for 2 h at 37°C. Nonadhered cells were washed off, and DC were then incubated with 100 μM biotinylated peptide for between 5 min and 1 h at 37°C. After incubation, cells were fixed with 4% formaldehyde in PBS for 15 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature.

For assessment of the amount of peptide taken up by DC, DC were next incubated with streptavidin–Alexa 488 (1/400 dilution in PBS) (Invitrogen, Cergy Pontoise, France) to detect the biotinylated peptide. In experiments to assess MHC class II colocalization, cells were incubated after fixation and permeabilization with anti-mouse I-Ap (anti-MHC class II; cross-reacts with I-As) (FITC labeled) (1/100 dilution in PBS) (BD Biosciences, Le Pont de Claix, France) overnight at 4°C and then with streptavidin–Cy3 (1/400 dilution in PBS) (Invitrogen) at room temperature for 45 min. After staining, cells were washed in PBS, and coverslips were mounted using Aquapolymount medium. Immunofluorescence staining was monitored with a laser scanning microscope (Leica SP5 II) equipped with a Leica HCX PL APO ×63 oil differential interference contrast immersion lens (numerical aperture 1.4-0.6).

Uptake of APL and S-palmAPL also was compared using flow cytometry. DC in suspension were incubated with 10 μM of the different biotinylated peptides for 10 min at 37°C. At the end of the incubation, cells were washed and incubated with anti-CD11c (APC labeled) and anti–I-Ap (FITC labeled) (1/300 dilution) (BD Biosciences) to detect DC. After washing, the cells were fixed and permeabilized with BD Cytofix/Cytoperm and incubated with PE–streptavidin to detect the biotinylated peptides. The cells were analyzed by flow cytometry using a FACSCalibur system (BD Biosciences). Samples were gated on the CD11c+/I-Ap+ population, and the mean fluorescence intensity of staining with PE–streptavidin was determined.

Statistics.

Most statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA). Normality tests were initially performed to determine whether the data were normally distributed. Differences between more than two groups were tested using ANOVA (for parametric data) or the Kruskal–Wallis test (for nonparametric data). If analysis of the group as a whole showed a significant difference (p < 0.05), then appropriate posttests (Dunnett’s test with ANOVA or Dunn’s multiple comparisons test with Kruskal–Wallis) were used to compare pairs of groups. For analysis of the course of EAE, multiple time point two-way ANOVA was used. For comparison of disease incidence in mice, χ2 analysis with Yates’ correction was used. The log-rank (Mantel–Cox) test was used to compare survival curves (showing percentage of mice free of EAE).

The APL A188 is derived from the encephalitogenic peptide PLP178–191 by substituting an alanine (A) for phenylalanine at position 188. A188 is not encephalitogenic in vivo and is able to protect against the induction of PLP178–191-induced EAE in SJL/J mice when it is used at a higher molar ratio than the inducing peptide (14). A188 and S-palmA188 were synthesized by solid phase-synthesis (Table I); the palmitic chain was linked via a thioester bond to Cys183 in the S-palmA188 peptide. We then compared the effects of A188 and S-palmA188. Initial studies showed that neither A188 nor S-palmA188 were encephalitogenic when the peptides (emulsified in CFA) were injected into SJL/J mice (Table II). Next, mice were coimmunized with mixtures of encephalitogenic PLP178–191, together with either A188 or S-palmA188 at various molar ratios, and followed for development of EAE. The protective effect of A188 was significantly enhanced by thiopalmitoylation, as evidence by decreased incidence of disease, increased mean day of onset, and decreased mean severity of EAE (Fig. 1, Table II). When S-palmA188 was injected at a ratio of 1:1 with PLP178–191, animals were completely protected from disease; in contrast, EAE in mice treated with nonpalmitoylated A188 at a 1:1 ratio with PLP178–191 did not differ significantly from mice injected with PLP178–191 alone (Table II). In addition, a substantially smaller quantity of S-palmAPL than A188 was required to induce the same clinical effect, with 1:0.1 PLP178–191:S-palmA188 treatment giving the same outcome as 1:5 PLP178–191:A188 (Fig. 1).

Table II.
The effect of A188 and S-palmA188 on induction of EAE with PLP178–191 and of Q144 and S-palmQ144 on induction of EAE with PLP139–151 in coimmunization experiments at different ratios
Immunized with (Molar Ratio)IncidenceDay of Onseta (Mean ± SE)Average Scoresb (Mean ± SE)Severity Scorec (Mean ± SE)Disease durationd (Mean ± SE)
PLP178–191 8/8 11.3 ± 0.5 2.8 ± 0.3 2.8 ± 0.3 6.4 ± 1.1 
A188 alone 0/4 — 
S-palmA188 0/4 — 
PLP178–191 + A188 (1:5) 5/12* 11.5 ± 0.4 1.0 ± 0.4** 2.8 ± 0.4 1.8 ± 0.9** 
PLP178–191 + A188 (1:1) 3/4 11.3 ± 0.7 1.9 ± 0.8 2.5 ± 0.9 3.3 ± 1.5 
PLP178–191 + S-palmA188 (1:1) 0/8* e 0*** 0e 0*** 
PLP178–191 + S-palmA188 (1:0.2) 3/7 14.7 ± 0.9* 0.5 ± 0.3** 1.2 ± 0.4 0.7 ± 0.4** 
PLP178–191 + S-palmA188 (1:0.1) 3/8 11.7 ± 0.2 0.9 ± 0.4** 2.3 ± 0.2 2.5 ± 1.5* 
PLP139–151 4/4 11.5 ± 0.9 4.3 ± 0.3 4.3 ± 0.3 8.3 ± 0.9 
PLP139–151 + Q144 (1:6) 3/4 11.3 ± 0.6 2.1 ± 0.7* 2.8 ± 0.4* 4.3 ± 1.5 
PLP139–151 + S-palmQ144 (1:6) 0/4* e 0*** 0e 0*** 
PLP139–151 + S-palmQ144 (1:1) 1/3 21e 0.7 ± 0.7** 2e 1.3 ± 1.3** 
Immunized with (Molar Ratio)IncidenceDay of Onseta (Mean ± SE)Average Scoresb (Mean ± SE)Severity Scorec (Mean ± SE)Disease durationd (Mean ± SE)
PLP178–191 8/8 11.3 ± 0.5 2.8 ± 0.3 2.8 ± 0.3 6.4 ± 1.1 
A188 alone 0/4 — 
S-palmA188 0/4 — 
PLP178–191 + A188 (1:5) 5/12* 11.5 ± 0.4 1.0 ± 0.4** 2.8 ± 0.4 1.8 ± 0.9** 
PLP178–191 + A188 (1:1) 3/4 11.3 ± 0.7 1.9 ± 0.8 2.5 ± 0.9 3.3 ± 1.5 
PLP178–191 + S-palmA188 (1:1) 0/8* e 0*** 0e 0*** 
PLP178–191 + S-palmA188 (1:0.2) 3/7 14.7 ± 0.9* 0.5 ± 0.3** 1.2 ± 0.4 0.7 ± 0.4** 
PLP178–191 + S-palmA188 (1:0.1) 3/8 11.7 ± 0.2 0.9 ± 0.4** 2.3 ± 0.2 2.5 ± 1.5* 
PLP139–151 4/4 11.5 ± 0.9 4.3 ± 0.3 4.3 ± 0.3 8.3 ± 0.9 
PLP139–151 + Q144 (1:6) 3/4 11.3 ± 0.6 2.1 ± 0.7* 2.8 ± 0.4* 4.3 ± 1.5 
PLP139–151 + S-palmQ144 (1:6) 0/4* e 0*** 0e 0*** 
PLP139–151 + S-palmQ144 (1:1) 1/3 21e 0.7 ± 0.7** 2e 1.3 ± 1.3** 
a

Average of the day postimmunization that first clinical signs were recorded in the mice that developed EAE.

b

Average of the scores of all mice.

c

Average of the scores of mice that developed EAE.

d

Average of the number of consecutive days an EAE score was recorded.

e

Statistical analyses could not be done for these groups because of fewer than three mice per group with EAE.

*p < 0.05, **p < 0.01, and ***p < 0.001 compared with mice immunized with PLP178–191 or PLP139–151 alone.

FIGURE 1.

(A) Clinical course of EAE in mice immunized with PLP178–191, PLP178–191 + A188 (at 1:5 ratio), or PLP178-191 + S-palmA188 (at 1:1 and 1:0.1 ratios). At 1:1 ratio, the S-palmA188 completely prevented onset of EAE (p < 0.0001 compared with PLP178–191 at days 11–17 and p < 0.01 at day 18). S-palmA188 at 1:0.1 ratio was as effective as A188 at 1:5 ratios; both of these were significantly less than the PLP178–191 group at days 12–18. (B) The percentage of mice free of EAE. All mice in the PLP178–191 group had developed EAE by day 14 after induction of EAE. All mice in the S-palmA188 (1:1) group remained free of disease, which was highly significantly different from the PLP178–191 group (p < 0.0001). Approximately 60% of the mice in both the A188 (1:5) and S-palmA188 (1:0.1) groups remained free of EAE (p values given next to these lines are for the comparison with the PLP178–191 group).

FIGURE 1.

(A) Clinical course of EAE in mice immunized with PLP178–191, PLP178–191 + A188 (at 1:5 ratio), or PLP178-191 + S-palmA188 (at 1:1 and 1:0.1 ratios). At 1:1 ratio, the S-palmA188 completely prevented onset of EAE (p < 0.0001 compared with PLP178–191 at days 11–17 and p < 0.01 at day 18). S-palmA188 at 1:0.1 ratio was as effective as A188 at 1:5 ratios; both of these were significantly less than the PLP178–191 group at days 12–18. (B) The percentage of mice free of EAE. All mice in the PLP178–191 group had developed EAE by day 14 after induction of EAE. All mice in the S-palmA188 (1:1) group remained free of disease, which was highly significantly different from the PLP178–191 group (p < 0.0001). Approximately 60% of the mice in both the A188 (1:5) and S-palmA188 (1:0.1) groups remained free of EAE (p values given next to these lines are for the comparison with the PLP178–191 group).

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To ensure that the protective effects observed were not restricted to a single APL, we also tested the APL Q144, derived from the encephalitogenic peptide PLP139–151 (Table I). Previous work has shown that Q144 induces a highly Th2 polarized response and is effective at inhibiting EAE induced by PLP139–151 at a 1:6 ratio of PLP139–151:Q144 (9). As shown in Table II, the protective effect of Q144 was also enhanced when the peptide was palmitoylated on Cys150.

We have previously shown that the APL A188 protects against EAE primarily via induction of A188-specific CD4+ Th0/Th2 cells (14). To determine whether a stronger A188-specific CD4+ T cell response was induced in mice immunized with the thiopalmitoylated peptide, we next compared the proliferation of CD4+ and CD8+ T cells from mice immunized with A188, S-palmA188, or PLP178–191 in response to PLP178–191 or A188. We have used A188 to stimulate T cells generated in mice immunized with either A188 or S-palmA188, as we have shown in a previous study that the lipid tail is cut off the peptide within the endosomes or lysosomes of the APCs, because of the presence of thioesterases in these organelles (5); thus, the T cells would recognize only the A188 portion of the molecule, irrespective of whether the APCs take up nonpalmitoylated peptide or thiopalmitoylated peptide.

LNC from mice immunized 10 d previously with A188, S-palmA188, or PLP178–191 were stained with CFSE and stimulated with Ag in vitro for 5 d. LNC were subsequently stained with anti-CD4 or anti-CD8 Abs and analyzed to assess cell proliferation. Very few proliferating CD8+ cells were detected (data not shown). CD4+ T cells proliferated in an Ag-specific manner (Table III). Most notably, the percentage of proliferating A188-specific T cells was twice as high in LNC from mice immunized with S-palmA188 (10.7 ± 2.6%) than in LNC from mice immunized with A188 (5.2 ± 1.5%), showing that thiopalmitoylation efficiently enhances the proliferation of CD4+ T cells. PLP178–191-specific T cells proliferated minimally following stimulation with A188, but proliferated strongly in response to PLP178–191. There were no significant differences in the ability of the LNC from mice immunized with A188 or S-palmA188 to proliferate in response to PLP178–191 or to the unrelated PLP139–151 peptide.

Table III.
Assessment of proliferation of Ag-primed CD4+ cells, as determined by CFSE and anti-CD4 Ab staining and flow cytometry
Ag-Primed LNCStimulating Aga% Dividing CD4+ Cells (Mean ± SE)bCell Division Index
A188 None 1.1 ± 0.2  
 A188 5.2 ± 1.5* 4.6 
 PLP178–191 1.4 ± 0.3 1.2 
 PLP139–151 1.4 ± 0.3 1.2 
S-palmA188 None 0.9 ± 0.2  
 A188 10.7 ± 2.6** 12.2 
 PLP178–191 1.6 ± 0.45 1.8 
 PLP139–151 2.6 ± 1.3 2.9 
PLP178–191 None 1.5 ± 0.5  
 A188 2.5 ± 0.8 1.7 
 PLP178–191 21.4 ± 8.3* 14.3 
 PLP139–151 2.9 ± 1.3 1.9 
Ag-Primed LNCStimulating Aga% Dividing CD4+ Cells (Mean ± SE)bCell Division Index
A188 None 1.1 ± 0.2  
 A188 5.2 ± 1.5* 4.6 
 PLP178–191 1.4 ± 0.3 1.2 
 PLP139–151 1.4 ± 0.3 1.2 
S-palmA188 None 0.9 ± 0.2  
 A188 10.7 ± 2.6** 12.2 
 PLP178–191 1.6 ± 0.45 1.8 
 PLP139–151 2.6 ± 1.3 2.9 
PLP178–191 None 1.5 ± 0.5  
 A188 2.5 ± 0.8 1.7 
 PLP178–191 21.4 ± 8.3* 14.3 
 PLP139–151 2.9 ± 1.3 1.9 
a

Con A was also used as a positive control in all assays (data not shown).

b

Results are the mean ± SE of three replicate experiments.

*p < 0.05, **p < 0.01 compared with no Ag control of LNC primed with the same Ag.

Previously, we have reported that A188-specific T cells have elevated levels of mRNA for the cytokines IL-4, IL-5, IL-10, and IFN-γ, compared with controls, and have speculated that the APL prevents EAE by inducing the production of Th0 and Th2 cytokines by A188-specific cells (14). Since the time when those experiments were done, Th17 cells have been shown to play an important role in the development of EAE (18). To evaluate the effect of thiopalmitoylation of A188 on the production of cytokines, we measured levels of three key cytokines (IFN-γ, IL-10, and IL-17A). Culture supernatants were collected from 72-h in vitro cultures (in the presence of no Ag, PLP178–191, or A188) of LNC from mice that had been immunized with PLP178–191, A188, or S-palmA188. Significant differences were seen among the different groups with respect to production of IFN-γ, IL-10, and IL-17A (Fig. 2).

FIGURE 2.

Production of cytokines (IFN-γ, IL-10, and IL-17) by LNC from mice immunized with PLP178–191, A188, or S-palmA188 following stimulation with PLP178–191 (black bars) or A188 (stippled bars), as indicated by fold change compared with unstimulated cells. Background levels of cytokines were as follows: IFN-γ, 37.5 ± 10.5 pg/ml; IL-10, 54.3 ± 17.6 pg/ml; and IL-17, 5.1 ± 0.9 pg/ml.

FIGURE 2.

Production of cytokines (IFN-γ, IL-10, and IL-17) by LNC from mice immunized with PLP178–191, A188, or S-palmA188 following stimulation with PLP178–191 (black bars) or A188 (stippled bars), as indicated by fold change compared with unstimulated cells. Background levels of cytokines were as follows: IFN-γ, 37.5 ± 10.5 pg/ml; IL-10, 54.3 ± 17.6 pg/ml; and IL-17, 5.1 ± 0.9 pg/ml.

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Following in vitro stimulation with PLP178–191, LNC from mice immunized with PLP178–191 showed a >500-fold increase in the amount of IL-17A produced and smaller fold changes in levels of IFN-γ and IL-10. After in vitro stimulation with A188, these same LNC increased production of IL-10 but showed minimal increases in levels of IFN-γ and IL-17. In contrast, LNC from mice immunized with A188 or S-palmA188 cells showed no increases in the amounts of IL-17 when stimulated in vitro with either A188 or PLP178–191. A188-specific and S-palmA188–specific LNC both increased production of IFN-γ in response to A188 stimulation but not when stimulated with PLP178–191. The most significant finding from this experiment was the greatly enhanced ability of S-palmA188–specific LNC to produce the anti-inflammatory cytokine IL-10 following stimulation with A188 (p < 0.001 compared with other groups).

Because S-palmA188 was able to induce production of significantly higher levels of the anti-inflammatory cytokine IL-10 than could A188, we next tested whether some of the effects of the S-palmA188 might be due to bystander suppression of proinflammatory responses in a non–Ag-specific manner. To test this, mice were immunized with 32 μmol PLP139–151 alone, 32 μmol PLP139–151 plus equimolar amount of A188, or 32 μmol PLP139–151 plus equimolar amount of S-palmA188. Mice were then followed for 35 d. All mice immunized with PLP139–151 developed EAE, although because the amount of PLP139–151 used to induce disease was less than half of the amount used for disease induction in the mice in Table II, the average day of onset of EAE was slightly later than that shown in Table II (Fig. 3A), and the mean severity was lower (2.8 ± 0.4). Three of four mice coimmunized with PLP139–151 and A188 also developed EAE with the same kinetics of disease (Fig. 3A). In contrast, only two of eight mice coimmunized with PLP139–151 and S-palmA188 developed EAE (Fig. 3A). In the three A188-coimmunized and 2 S-palmA188–coimmunized mice that did develop EAE, disease severity was not significantly less than that seen in the mice immunized with PLP139–151 alone. These results strongly suggest that a bystander response induced by S-palmA188 was sufficient to overcome the encephalitogenicity of PLP139–151.

FIGURE 3.

S-palmA188 can induce non–Ag-specific protection in the coimmunization model, probably through induction of a Th0-like response. (A) Mice were immunized with PLP139–151 alone or coimmunized with PLP139–151 together with A188 or S-palmA188. The percentage of mice free of EAE is shown. All mice immunized with PLP139–151 alone (n = 8) developed EAE, as did three of four mice coimmunized with PLP139–151 and A188. In contrast, only two of eight mice coimmunized with PLP139–151 and S-palmA188 developed EAE, which was significantly different to the control group (p = 0.0002 by the log-rank [Mantel–Cox] test). (B) At day 36 after the commencement of the coimmunization experiment, LNC were removed from each mouse and activated in vitro with no Ag, PLP139–151, or A188. After 72 h, supernatants from each culture were harvested and subsequently tested for production of IL-10 and IFN-γ in CBA assays. The levels of cytokines (mean ± SE for each group of mice) are expressed as picograms per milliliter.

FIGURE 3.

S-palmA188 can induce non–Ag-specific protection in the coimmunization model, probably through induction of a Th0-like response. (A) Mice were immunized with PLP139–151 alone or coimmunized with PLP139–151 together with A188 or S-palmA188. The percentage of mice free of EAE is shown. All mice immunized with PLP139–151 alone (n = 8) developed EAE, as did three of four mice coimmunized with PLP139–151 and A188. In contrast, only two of eight mice coimmunized with PLP139–151 and S-palmA188 developed EAE, which was significantly different to the control group (p = 0.0002 by the log-rank [Mantel–Cox] test). (B) At day 36 after the commencement of the coimmunization experiment, LNC were removed from each mouse and activated in vitro with no Ag, PLP139–151, or A188. After 72 h, supernatants from each culture were harvested and subsequently tested for production of IL-10 and IFN-γ in CBA assays. The levels of cytokines (mean ± SE for each group of mice) are expressed as picograms per milliliter.

Close modal

To investigate whether this non–Ag-specific protection might be due to the production of IL-10 and/or IFN-γ, LNC were removed from the above mice on day 35 after immunization. At the time when LNC were collected, all mice were in the recovery or remission phase of disease. Cultures were set up from each individual mouse’s LNC to assess the production of IL-10 and IFN-γ upon stimulation of the LNC with no Ag, PLP139–151, or A188. After 3-d in vitro stimulation, culture supernatants were harvested, and the levels of IL-10 and IFN-γ in the supernatants were assessed using CBA assays. All mice were found to make a low level of IL-10 upon stimulation with PLP139–151 (Fig. 3B). However, only LNC from mice coimmunized with either A188 or S-palmA188 made significantly increased amounts of IL-10 upon stimulation with A188 (Fig. 3B). A188-coimmunized mice had a 12-fold increase in IL-10 levels, compared with the no Ag group. In S-palmA188–coimmunized mice, there was a 19.4-fold increase in IL-10 levels. Significant fold changes in levels of IFN-γ were also seen in the mice coimmunized with either A188 or S-palmA188 (3.6-fold increase in each, compared with no Ag group). These results support the proposition that the production of significant amounts of IL-10 by cells responding to S-palmA188 can act nonspecifically to decrease the encephalitogenic response normally induced by PLP139–151. Because A188 coimmunized mice also produced reasonable amounts of IL-10, it may be that there is a threshold amount of IL-10 required to induce protection from EAE, which was not quite reached in the A188-treated animals.

Another potential reason for the enhanced effectiveness of S-palmA188 in vivo could relate to its bioavailability. We therefore next investigated the stability of A188 and S-palmA188 in the presence of serum. For this purpose, both peptides were incubated in phosphate buffer in the presence of 10% FCS, and the progress of the digestion was followed by HPLC. As shown in Fig. 4, the S-palm peptide exhibited greater stability than the nonpalmitoylated peptide, because most of the A188 was digested after 3 h, whereas 85% of S-palmA188 remained undigested at that time point. The half-life of S-palmA188 was significantly longer (>850 min) than that of A188 (50 min), which would enhance the bioavailability of S-palmA188.

FIGURE 4.

Kinetics of digestion of A188 and S-palmA188 in the presence of serum. The dotted lines show the half-lives (t1/2) of each peptide.

FIGURE 4.

Kinetics of digestion of A188 and S-palmA188 in the presence of serum. The dotted lines show the half-lives (t1/2) of each peptide.

Close modal

DC are the professional APC of the immune system. Previously, we have found that thiopalmitoylated encephalitogenic peptides are not only taken up more efficiently by another type of APC (macrophages) than are the same nonpalmitoylated peptides, but are also channeled into the MHC class II presentation pathway, and we have proposed that this underlies their improved immunogenicity and the enhancement of CD4+ T cell responses (4, 5). In this study, we sought to confirm whether S-palmAPL was taken up more efficiently than nonpalmitoylated APL by DCs.

DC were purified from spleens of SJL/J mice and incubated for 10 min in the presence of biotinylated S-palmA188 or nonpalmitoylated A188 (Table I). PE-conjugated streptavidin was used to detect the peptide taken up by these cells by flow cytometric analysis. Thiopalmitoylation enhanced the uptake of APL by DCs, as indicated by higher levels of PE staining (Fig. 5A). This was further confirmed using confocal microscopy on purified splenic DCs incubated with the APL and S-palmAPL for different times (Fig. 5B–F). S-palmA188 could be easily detected at the cellular membrane after 5 min (Fig. 5B), and after just 15 min, the translocated peptide showed a punctate fluorescence staining pattern, indicative of localization in discrete vesicular compartments and suggestive of an endocytic pathway of internalization (Fig. 5C–E). In contrast, even after 60 min, hardly any non-palmA188 peptide had been taken up into DC (Fig. 5F). These results confirm that the palmitic chain markedly enhances the efficiency of peptide translocation into DC.

FIGURE 5.

The uptake of A188 and S-palmA188 peptide by splenic DCs. (A) Peptide uptake after 10 min indicated by fluorescent intensity measured via flow cytometry. (BF) Confocal microscopy analysis of uptake of biotinylated peptides (stained green). (B–E) uptake of S-palmA188 after the indicated times or (F) uptake of A188 after 60 min. (G) Colocalization of MHC class II and peptide indicated by merging (yellow) of MHC class II staining (green) and peptide staining (red). Scale bar, 5 μm.

FIGURE 5.

The uptake of A188 and S-palmA188 peptide by splenic DCs. (A) Peptide uptake after 10 min indicated by fluorescent intensity measured via flow cytometry. (BF) Confocal microscopy analysis of uptake of biotinylated peptides (stained green). (B–E) uptake of S-palmA188 after the indicated times or (F) uptake of A188 after 60 min. (G) Colocalization of MHC class II and peptide indicated by merging (yellow) of MHC class II staining (green) and peptide staining (red). Scale bar, 5 μm.

Close modal

We have previously demonstrated with encephalitogenic thiopalmitoylated peptides that the thioester bond between the lipid and peptide can be broken down in endosomes and lysosomes (part of the MHC class II presentation pathway and which contain thioesterases), thereby effectively “stranding” the peptide in these organelles, and making it much more likely that they will be presented by MHC class II molecules (5). To assess whether this same mechanism was likely to be occurring with the S-palmA188 peptide, we investigated whether there was colocalization of biotinylated peptide and MHC class II in DC that has been incubated with the biotinylated peptide for 60 min. Most of the biotinylated peptide (detected using streptavidin–Cy3) colocalized with MHC class II (Fig. 5G). These results confirm that the thioester bond between lipid and peptide helps to channel the peptide into the MHC class II presentation pathway, because of the presence of thioesterases in these organelles, thereby inducing a CD4+ T cell response, as we have previously found with encepalitogenic peptides in macrophages (5).

In the current study, we have shown that thiopalmitoylation of APLs can enhance their protective properties. Using a coimmunization protocol, we showed that thiopalmitoylation greatly enhanced the prophylactic abilities of the A188 APL (Fig. 1, Table II), with a 50-fold lesser quantity of S-palmA188 needed to induce the same protective effects as nonpalmitoylated A188. The complementary data obtained from S-palmQ144 show that the enhancement of the protective effects afforded by thiopalmitoylation is not exclusive to one APL but can be generalized to other APLs. These results support the idea that thiopalmitoylation of a peptide enhances its innate properties; thus, thiopalmitoylation of a peptide that has the potential to be pathogenic can enhance pathogenicity (as we have previously shown in EAE (2, 5) and experimental autoimmune neuritis (3)), whereas thiopalmitoylation of a peptide that has the potential to protect against disease can enhance the efficacy of the therapeutic effect. This could have potential applicability in vaccine development, providing a simple means to increase MHC class II–restricted T cell responses.

There appear to be several mechanisms that are operative in the enhancement of immunogenicity of the A188 APL by thiopalmitoylation. First, thiopalmitoylation greatly enhanced the stability and longevity of A188 peptide in serum, with S-palmA188 having a half-life > 15 times that of the nonpalmitoylated APL in the presence of serum (880 versus ∼50 min, respectively). This could be due to formation of micelle-like aggregations or particles, as has previously been reported for N-palmitoylated peptides (19, 20). The characteristic of enhanced stability in serum is yet to be confirmed for other S-palm peptides; however, the increase in bioavailability is almost certainly one element important for the enhanced immunological activity and in vivo effects of S-palmA188.

Second, uptake of S-palmA188 compared with A188 into the class II MHC presentation pathway of DC was greatly enhanced. Transient thioacylation, particularly by attachment of myristic acid (C14) or palmitic acid (C16), is a common mechanism that cells use to move molecules through membranes; this can occur via nonphagocytic mechanisms, endocytosis, or patocytsis (reviewed in Ref. 21). Our previous work suggests that thiopalmitoylation of PLP peptides allows uptake via nonphagocytic mechanisms (5). Furthermore, using S-palm peptides in which the palmitic acid is replaced by a fluorescent lipid analog, we have previously shown that the lipid moiety is cleaved from the peptide within endosomes, thereby stranding the peptide moiety in the endosome (5); this would account for the preference for the class II MHC pathway, because the endosomes are part of this pathway.

Last, S-palmA188–primed cells, compared with those primed by A188, showed an enhanced ability to proliferate (Table III) and to produce the anti-inflammatory cytokine IL-10 (Fig. 2) in the presence of APL stimulation; this can explain the increased efficacy of S-palmA188 in vivo compared with nonconjugated A188. Interestingly, both A188- and S-palmA188–primed cells produced copious amounts of IFN-γ, a cytokine often regarded as proinflammatory; however, it is known that IFN-γ can inhibit Th17 responses (22, 23), and furthermore, several studies have reported more severe and chronic disease in anti–IFN-γ Ab–treated (24) and IFN-γ gene knockout (25) EAE models. Taken together, these findings support the idea that IFN-γ can cause downregulation of pathogenic Th17-mediated disease (26). It is also of interest to note that development of mucosal tolerance to another myelin Ag, myelin basic protein, recently has been reported to be mediated by a Th0 cell population producing both IFN-γ and IL-10 (27), similar to the phenotype of the S-palmA188–specific cells reported in this study.

Because of the induction of copious amounts of IL-10 in mice immunized with S-palmA188, we also tested the potential of S-palmA188 to protect against induction of EAE with an unrelated Ag. As shown in Fig. 3, coimmunization with S-palmA188, but not A188, effectively protected against disease induced by PLP139–151. This protection correlated with enhanced production of IL-10 in the mice coimmunized with PLP139–151 and S-palmA188. There was also an elevated level of IL-10 produced in mice coimmunized with PLP139–151 and A188, although not to extent of that induced by S-palmA188. These results suggest that S-palmAPLs could potentially have both specific and nonspecific bystander therapeutic effects: in a therapeutic setting, this could be a distinct advantage.

A small number of human APL trials have been undertaken in patients with autoimmune disease (2832); they have not been very successful thus far, although this has been due largely to trial design issues such as poor selection of APLs (using APLs that were capable of acting as full agonists for some patient T cell clones (33, 34)) and of patients (not screening patients to ensure that they carried the specific HLA molecules for which the APLs were designed). However, part of the lack of success of the human APL trials also stems from the poor in vivo stability and low immunogenicity of peptides, and the severe injection-site reactions that developed when very large amounts of peptide were administered to try to overcome these issues. The findings in the current study suggest that S-palmAPL may have significant advantages over APLs in future human trials, given their markedly increased stability and immunogenicity, provided APL- and patient-selection issues are adequately addressed.

Thiopalmitoylation of other types of peptide vaccines also may help to enhance CD4+ T cell–mediated prophylactic or therapeutic effects. For example, current studies are trying to find a way to enhance CD4+ T cell responses to cancer vaccines, so as to promote more robust priming and long-term protective CD8+ T cell responses (35). Thiopalmitoylation of peptides may be one way to achieve this. The protective effects of the S-palmAPLs do not appear to be dependent on the position in the peptide chain of the cysteine residue to which the palmitic chain is linked via a thioester bond, because this was the second residue for PLP139–151 and the sixth residue for PLP178–191. If a peptide does not contain a cysteine in its sequence, it may be possible to replace an existing serine with a cysteine without affecting the biological activity (3), or to add a cysteine at the N or C terminus of the peptide.

Taken together, our results suggest that the strategy of thiopalmitoylation of a peptide has great potential to enhance CD4+ T cell responses to that peptide. The lipid group does not change the type of response that is induced; it merely enhances the type of response that the peptide has the capacity to induce. We provide evidence that S-palmAPL can be highly efficient at a much lower dose than the non-palm APL. This strategy could effectively and safely enhance APL therapy for many autoimmune and inflammatory diseases, including MS, diabetes type 1, rheumatoid arthritis and myasthenia gravis. Thiopalmitoylated peptides also have the potential to enhance immunogenicity of peptide vaccines that aim to induce a CD4+ T cell response.

We thank Dr. Jean-Marc Strub (Laboratoire de Spectométrie de Masse Bioorganique, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France) for the mass spectrometry analysis of the peptides, Dr. Estelle Hess (Immunopathologie et Chimie Thérapeutique, Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France) for her help in flow cytometry analysis, and Dr. Susana Brun for critical reading of the manuscript.

This work was supported by a grant from Multiple Sclerosis Research Australia.

Abbreviations used in this article:

A

alanine

APL

altered peptide ligand

CBA

cytokine bead array

EAE

experimental autoimmune encephalomyelitis

LNC

lymph node cell

MS

multiple sclerosis

PLP

proteolipid protein

Q

glutamine.

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The authors have no financial conflicts of interest.