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. 2012 Dec 13;138(1):68–75. doi: 10.1111/imm.12019

The choice of adjuvant determines the cytokine profile of T cells in proteoglycan-induced arthritis but does not influence disease severity

Jeroen N Stoop 1,*, Christopher A Tibbitt 1, Willem Eden 2, John H Robinson 1, Catharien M U Hilkens 1
PMCID: PMC3533702  PMID: 23077978

Abstract

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) -dependent disease that is induced with antigen in complete Freund's adjuvant, whereas PGIA is Th1-mediated and is induced using antigen in dimethyldioctadecyl-ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T-cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan-T-cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin-17 (IL-17) produced by T cells isolated from CFA-immunized mice after antigen-specific stimulation were not significantly different from those found in DDA-immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL-17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity.

Keywords: arthritis, CD4+ T cells, interferon-γ and adjuvant, interleukin-17

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a chronic inflammation of synovial joints, causing cartilage resorption and bone destruction.1 The synovial tissue of RA patients is heavily infiltrated with T cells2 and T-cell-derived cytokines are implicated in joint destruction.3 Both T helper type 1 (Th1) and Th17 cells have been associated with RA pathogenesis.4,5 The Th1 cells are thought to play an important role by providing help for B cells and activating monocytes within joints.1 The Th17 cells are a source of interleukin-17 (IL-17), which can mediate joint damage by inducing the production of pro-inflammatory cytokines, matrix metalloproteinases and the promotion of osteoclastogenesis.6 Although an increased proportion of Th17 cells can be found in patients with RA, several groups have shown that there are very few Th17 cells in RA synovium and that most of the IL-17-positive cells are mast cells.5,7,8

Proteoglycan-induced arthritis (PGIA) and collagen-induced arthritis (CIA) are two mouse models used to study inflammatory arthritis. Both models show many of the features of RA. PGIA has been studied in BALB/c mice following immunization with proteoglycan aggrecan (PG) using either complete Freund's adjuvant (CFA) containing heat-killed Mycobacterium tuberculosis organisms or, in more recent reports, with dimethyldioctadecyl ammonium bromide (DDA) as adjuvant.9,10 In studies using DDA as an adjuvant, Th1 cells were shown to predominate as the pathogenic T-cell population in PGIA. The PGIA could also be induced in IL-17 knockout mice, indicating that IL-17 and Th17 cells are not required for destructive joint inflammation in mice.9 However, in the absence of interferon-γ (IFN-γ), IL-17 has been shown to contribute to pathogenicity.10 The T-cell cytokine profile in mice in which PGIA was induced with CFA has not been studied in detail. It has been shown that PG-specific T-cell receptor transgenic (PG-TCRtg) BALB/c mice develop PGIA more rapidly after PG immunization and show a more severe arthritis compared with wild-type BALB/c mice.11 These PG-TCRtg mice are therefore a useful tool with which to study the activation and differentiation of naive joint antigen-specific T cells.

Collagen-induced arthritis is the most frequently used animal model for arthritis; it is induced in DBA/1 or C57BL/6 mice by immunization with type II collagen (CII) in CFA,12 and has been shown to be Th17-dependent.13 This poses a potential problem. Both PGIA and CIA are considered to be animal models for RA and are used to test potential therapeutic strategies; however, whereas CIA is thought to be Th17 dependent, PGIA is Th1 dependent. Both of these models depend on the use of adjuvants (e.g. DDA or CFA). CFA is a molecularly poorly defined adjuvant, in which the main active ingredients are heat-killed bacteria. Although DDA is molecularly defined, its mechanism of action has not been studied in detail. Nevertheless, it has been shown that immunizing BALB/c mice with PG in DDA results in high levels of PG-specific IFN-γ production.14 However, little is known about the effect of DDA on Th17 responses.

The three main factors that could be responsible for the observed differences in T-cell polarization between the CIA and PGIA models are the mouse strain, the antigen and the adjuvant. We hypothesize that the type of adjuvant plays an important role in determining the type of pathogenic T-cell response that predominates and that disease induction with CFA will result in more Th17 cells. Therefore, we compared the in vivo effect of CFA and DDA in a single arthritis model (PGIA in PG-TCRtg mice) and show that the choice of adjuvant affects the relative proportions of Th1 and Th17 cells, but not the level of IL-17 or IFN-γ secreted in response to antigen or the disease severity.

Materials and methods

Induction and monitoring of arthritis

Male DBA/1 mice were purchased from Harlan Laboratories, Bicester, UK, and 5/4E8-TCR-Tg mice, transgenic for a Vβ4/Vα1.1 TCR specific for the major arthritogenic CD4 T-cell epitope of PG aggrecan 70ATEGRVRVNSAYQDK84 (PG-TCRtg) were bred in-house as described earlier.11 Experiments were performed under the terms of the Animals (Scientific Procedures) Act of 1986 and were authorized by the Home Secretary, Home Office, UK.

Proteoglycan-induced arthritis was induced in 12-week old PG-TCRtg mice by subcutaneous injection at the tail base with 100 μg bovine PG, prepared as described elsewhere,15 emulsified in CFA containing 1 mg/ml M. tuberculosis (Difco, Detroit, MI) on day 0, followed by a subcutaneous boost with 100 μg PG in incomplete Freund's adjuvant (IFA; Sigma-Aldrich, Poole UK) on day 21. Alternatively, mice were immunized by intraperitoneal injection with 100 μg PG emulsified in DDA (Sigma-Aldrich) on day 0, and again on day 21. CIA was induced in 8- to 10-week-old DBA/1 mice on day 0 by subcutaneous injection at the tail base with 100 μg bovine type II collagen (CII; Chondrex, Redmond, WA) emulsified in CFA. On day 21 mice received a subcutaneous boost with 100 μg bovine CII in IFA, followed by intraperitoneal injection of 25 μg ultra-pure lipopolysaccharide (InvivoGen, Toulouse, France) on day 24, to synchronize arthritis onset. In all cases, arthritis severity was monitored using a clinical score: 0 = no signs of inflammation, 1 = erythema or swelling, 2 = both erythema and swelling, and 3 = erythema and severe swelling, as described earlier.16 The clinical score per mouse was the cumulative value for all paws. The thickness of the feet (swelling) was measured with a spring-loaded Oditest caliper (Kroeplin GmbH, Schüchtern, Germany).

Generation of bone-marrow-derived dendritic cells

Cells were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (PAA Laboratories, Pasching, Austria), 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μm 2-mercaptoethanol (all from Sigma-Aldrich) at 37° with 5% CO2. Dendritic cells (DC) were generated as described previously.17 Briefly, bone marrow from the femurs and tibias of mice was cultured for 10 days in medium containing 20 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ). Cultures were refreshed on days 3, 6 and 8 with medium supplemented with GM-CSF. On day 10, DC were matured with 0·1 μg/ml lipopolysaccharide (Sigma-Aldrich) for 16 hr.

Flow cytometry

Fluorescent antibody labelling of cells was performed in PBS containing 0·5% BSA. FoxP3 labelling was performed according to the manufacturer's instructions. Briefly, cells were labelled with anti-CD4-Peridinin chlorophyll protein-Cy5.5 (PerCP-Cy5.5; RM4-5; BD Pharmingen, San Diego, CA) and anti-CD25-Alexa Fluor 488 (M-A251; eBioscience, San Diego, CA). The cells were fixed and permeabilized with Fix/Perm buffer and permeabilization buffer (eBioscience). Anti-FoxP3-APC (FJK-16a; eBioscience) and 4% rat serum (Sigma-Aldrich) were added during permeabilization.

For intracellular cytokine staining, cells were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (PAA Laboratories), 2 mm glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μm 2-mercaptoethanol (all from Sigma-Aldrich). For polyclonal stimulation cells were stimulated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) for 5 hr. Brefeldin A (10 μg/ml; Sigma-Aldrich) was added to the cells after 1 hr of stimulation. For antigen-specific intracellular cytokine staining, 1 × 106 splenocytes were co-cultured with 50 000 matured bone-marrow-derived DC (BMDC) in 24-well plates, in the absence or presence of 2 μm of the b84-103 proteoglycan peptide. These were incubated for 1 hr before the addition of 10 μg/ml Brefeldin A, with a further incubation for 20 hr at 37°, 5% CO2.

Cell surface labelling was performed with anti-CD4-PerCP-Cy5.5 and anti-CD8a-Pacific Blue (53-6.7; BD Pharmingen). Subsequently, cells were fixed and permeabilized as described above. Intracellular staining was performed in the presence of 4% rat serum using anti-IFN-γ-FITC (XMG1.2) and anti-IL-17-phycoerythrin (TC11-1H10.1; both from BD Pharmingen). Cells were acquired on a FACScanto or an LSR II (Beckton Dickinson, San Jose, CA) and data were analysed using Venturi One software (Applied Cytometry, Sheffield, UK).

Proliferation assays and ELISA

Spleen cells from arthritic mice were harvested after disease onset and were cultured in triplicate at a concentration of 3 × 105 cells per well in 200 μl RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (PAA Laboratories), 2 mm glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μm 2-mercaptoethanol (all from Sigma-Aldrich) at 37° with 5% CO2. Cells were stimulated with CD3/CD28 Dynal expander beads (Invitrogen Ltd., Paisley, UK), 2 μm b84-103 PG peptide (JPT peptide Technologies, Berlin, Germany), or 5 μg/ml tuberculin purified protein derivative (PPD; Statens Serum Institute, Copenhagen, Denmark), or were left unstimulated for 96 hr. After 80 hr, 100 μl supernatant was harvested and 10 kBq [3H]thymidine (specific activity; 74·0 GBq/mmol; Perkin Elmer, Shelton, CT) was added per well for the last 16 hr. Radioactivity was quantified using a beta scintillation counter (Perkin-Elmer Microbeta TriLux; Perkin Elmer, Waltham, MA). Sandwich ELISA with duo sets (R&D Systems, Abingdon, UK) was performed on supernatants to quantify IFN-γ and IL-17 levels. The ELISA were performed according to the manufacturer's instructions.

Statistics

Statistical analysis was performed using Mann–Whitney U-test in Prism 5 (Graphpad Software, La Jolla, CA).

Results

A higher proportion of Th1 cells in mice with PGIA contrasts with a higher proportion of Th17 cells in mice with CIA

To confirm the current consensus in literature that CIA is a Th17-mediated disease and PGIA is a Th1-mediated disease, we compared both models. We induced PGIA in PG-TCRtg mice by immunization with PG emulsified in DDA, as well as inducing CIA in DBA/1 mice by immunization with CII emulsified in CFA. After disease onset, when all mice had reached a clinical score of 3 or above, spleens were removed and the percentages of Th1 and Th17 cells were determined by intracellular cytokine staining after PMA/ionomycin stimulation. A higher proportion of IFN-γ-producing CD4+ T cells was found in mice with PGIA than with CIA. Conversely, a higher proportion of IL-17-secreting CD4+ T-cells was present in mice with CIA than in mice with PGIA, confirming that the Th1/Th17 ratio was higher in PGIA mice compared with CIA mice (Fig. 1).

Figure 1.

Figure 1

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Induction of arthritis and T helper type 1 (Th1)/Th17 cells in mice immunized with dimethyldioctadecyl-ammonium bromide (DDA) or complete Freund's adjuvant (CFA). Mice were immunized with proteoglycan (PG) in DDA (PG-TCRtg mice; squares) or type II collagen (CII) in CFA (DBA/1 mice; triangles). After the onset of PG-induced arthritis (PGIA) or collagen-induced arthritis (CIA), spleen cells were stimulated with PMA/ionomycin and the proportions of CD4+ T cells producing interferon-γ (IFN-γ) or interleukin-17 (IL-17) were determined by flow cytometry.

PGIA incidence or severity is unaffected by the choice of adjuvant

To compare the capacity of different adjuvants to induce PGIA, we immunized PG-TCRtg mice with PG in either CFA or in DDA. No difference was observed in the incidence of arthritis, and all mice had a clinical score of at least 3 within 30 days (Fig. 2a). Also, the choice of adjuvant did not affect the arthritis severity: no differences in clinical scores (Fig. 2b,c) or paw thickness (data not shown) were observed between the two groups. Mice that were sham-immunized with PBS in either DDA or CFA did not develop arthritis (data not shown).

Figure 2.

Figure 2

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Proteoglycan-specific T-cell receptor transgenic (PG-TCRtg) mice were immunized with either PG in dimethyldioctadecyl-ammonium bromide (DDA) (squares) or PG in complete Freund's adjuvant (CFA) (triangles). (a) Incidence of arthritis (clinical score of 3 or above). (b) Arthritis severity as the mean clinical score (± SEM). (c) The clinical score at the end of follow-up. Symbols in scatter plots represent individual mice and bars represent the median value in a group (n = 15).

A higher proportion of Th17 cells in PG/CFA-immunized mice contrasts with a higher proportion of Th1 cells in PG/DDA-immunized mice

We next compared the presence of relevant T-cell subsets in arthritic PG-TCRtg mice that had been immunized with PG in either DDA or CFA. No differences in the total spleen size (data not shown) or the proportion of CD4+ T cells, CD8+ T cells or FoxP3+ regulatory T cells were observed between DDA-immunized and CFA-immunized mice (Fig. 3a).

Figure 3.

Figure 3

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Proteoglycan-specific T-cell receptor transgenic (PG-TCRtg) mice were immunized with either PG in dimethyldioctadecyl-ammonium bromide (DDA) (squares) or PG in complete Freund's adjuvant (CFA) (triangles). After onset of PG-induced arthritis (PGIA), spleen cells were harvested and analysed by flow cytometry. For (b) and (c), harvested spleen cells were stimulated for 5 hr with PMA/ionomycin before analysis by flow cytometry. (a) Proportions of CD4+ T cells, CD8+ T cells, and FoxP3+ regulatory T cells. (b) Proportions of splenic CD4+ T cells producing interferon-γ (IFN-γ) and interleukin-17 (IL-17) and the T helper type 1 (Th1)/Th17 ratio. (c) Proportion of IFN-γ-producing CD8+ T cells. Symbols in scatter plots represent individual mice and bars represent median values in a group. Statistical analysis was performed using Mann–Whitney U-test (n = 12; *P < 0·05).

To determine the proportion of Th1 and Th17 cells, spleen cells were re-stimulated with PMA and ionomycin and cytokine production was studied by intracellular cytokine staining. Mice immunized with PG-in-DDA showed a higher proportion of IFN-γ-producing CD4+ T cells, whereas mice that were immunized with PG-in-CFA had a higher proportion of IL-17-secreting CD4+ T cells, resulting in a higher Th1/Th17 ratio in DDA-immunized mice (Fig. 3b). No difference was observed in the proportion of IFN-γ-producing CD8+ T cells (Fig. 3c).

PG/CFA-immunized mice have a higher proportion of b84-103-specific Th17 cells

To study the proportion of aggrecan b84-103-specific Th1 and Th17 cells, spleen cells from either PG-in-DDA- or PG-in-CFA-immunized mice were stimulated with peptide- pulsed BMDC and the cytokine production profile was determined by flow cytometry. A representative example of the intracellular cytokine staining is depicted in Fig. 4a. The PG-in-CFA-immunized mice had a higher proportion of Th17 cells than the PG-in-DDA-immunized mice and as a result, PG-in-CFA-immunized mice have a lower Th1/Th17 ratio (Fig. 4b). Together these data indicate that the use of CFA as an adjuvant results in the induction of a higher proportion of Th17 cells.

Figure 4.

Figure 4

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Proteoglycan-specific T-cell receptor transgenic (PG-TCRtg) mice were immunized with either PG in dimethyldioctadecyl-ammonium bromide (DDA) (squares) or PG in complete Freund's adjuvant (CFA) (triangles). Spleen cells were harvested, re-stimulated with b84-103 peptide and analysed by intracellular cytokine staining. (a) Representative example of an intracellular cytokine staining on spleen cells from a PG-in-DDA-immunized and a PG-in-CFA-immunized mouse. The CD4+ Vβ4+ cells are depicted in the dotplots. (b) Proportions of splenic CD4+ Vβ4+ T cells producing interferon-γ (IFN-γ) and interleukin-17 (IL-17) and the T helper type 1 ( Th1)/Th17 ratio after antigen-specific stimulation. Symbols in scatter plots represent individual mice and bars represent median values in a group. Statistical analysis was performed using Mann–Whitney U-test (n = 5; *P < 0·05).

PG-specific IL-17 and IFN-γ secretion in mice immunized in DDA and CFA are similar

To assess whether there were any differences in the PG-specific T-cell proliferation and cytokine secretion in PG-TCRtg mice immunized with PG in either DDA or CFA, spleen cells from arthritic mice were re-stimulated with the b84-103 peptide to determine proliferation and cytokine production. No differences in b84-103-specific proliferation between mice immunized with PG in DDA or CFA were observed (Fig. 5a). In contrast to the adjuvant-dependent Th1/Th17 polarization data obtained by intracellular cytokine analysis, no differences were observed in the b84-103-specific cytokine responses between mice immunized with PG/DDA and PG/CFA; similar levels of IFN-γ and IL-17 in culture supernatants were found (Fig. 5b).

Figure 5.

Figure 5

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Proteoglycan-specific T-cell receptor transgenic (PG-TCRtg) mice were immunized with either PG in dimethyldioctadecyl-ammonium bromide (DDA) (squares) or PG in complete Freund's adjuvant (CFA) (triangles). (a) Proliferation of spleen cells re-stimulated for 96 hr with 1 μm b84-103 peptide or CD3/CD28 beads, determined by [3H]thymidine incorporation (n = 12 mice). (b) Interferon-γ (IFN-γ) and interleukin-17 (IL-17) production of b84-103 peptide-stimulated spleen cells, determined by ELISA (n = 12 mice). (c) IFN-γ and IL-17 production by CD3/CD28-stimulated spleen cells, determined by ELISA (n = 12). (d) IL-17 and IFN-γ production of purified protein derivative (PPD) -stimulated spleen cells, determined by ELISA (n = 9 mice). Symbols represent individual mice and bars represent median percentages. Statistical analysis was performed using Mann–Whitney U-test (*P < 0·05).

Spleen cells from DDA-immunized mice produced higher levels of IFN-γ after polyclonal (CD3/CD28 bead) stimulation (Fig. 5c), consistent with data from intracellular cytokine staining experiments with PMA/ionomycin (Fig. 3b). However, no significant difference was seen in IL-17 production after CD3/CD28 bead stimulation (Fig. 5c). Hence, the CD3/CD28-induced IFN-γ secretion determined by ELISA followed the same trend as the intracellular cytokine data; however, the IL-17 secretion did not. Of note, no Th1 or Th17 polarization was observed in mice that were injected with DDA or CFA without PG, respectively, which also did not develop arthritis (data not shown).

We could detect T-cell responses against PPD, a constituent of M. tuberculosis. Interestingly, spleen cells from four out of nine PG/CFA-immunized mice produced IL-17 after re-stimulation with PPD (Fig. 5d). Cells from two of the four mice that produced IL-17 also produced IFN-γ (Fig. 5d). Hence, immunization of PG in CFA not only gave rise to PG-specific T-cell responses but could also induce T-cell responses against antigens present in CFA.

Discussion

CD4+ T cells are categorized based on patterns of cytokine production and the expression of certain transcription factors. Th1 cells are characterized by the expression of T-bet and production of IFN-γ whereas Th17 cells express RORγt and produce IL-17, IL-21 and IL-22 (reviewed in ref. 18).

T cells are thought to be important in the pathogenesis of RA.2 However, there is discussion in the literature on the role of Th17 cells in RA. An increased proportion of Th17 cells can be found in patients with RA, but most of the IL-17-positive cells in the synovium are mast cells.5,7,8 In mouse models the role of Th1 and Th17 cells is much more defined. PGIA and CIA are two well-established mouse models of inflammatory arthritis that are thought to be driven by Th1 and Th17 cells, respectively.9,13 Our study is the first direct comparison of the Th1/Th17 ratio in mice with PGIA and CIA in the same laboratory setting. We confirm that mice with PGIA show a higher Th1/Th17 ratio compared with mice with CIA. As both PGIA and CIA are used as experimental models to understand the pathogenesis of RA and to test potential therapeutic strategies, it is important to understand the cause of this discrepancy in cytokine production by T cells between these two models.

Focusing on the role of adjuvant in influencing T-cell polarization in arthritis models, the cytokine profile of T cells was examined in a single arthritis model where only the adjuvant was altered. Our finding that DDA induces a Th1 bias whereas CFA polarizes more towards Th17 indicates that the type of adjuvant contributes to the observed differences in T-cell polarization in CIA versus PGIA. We did not detect a difference in the clinical score between mice immunized with PG-in-CFA and PG-in-DDA, whereas there is a difference in the Th1/Th17 ratio. Doodes et al.10 have shown that in the absence of IFN-γ, IL-17 plays a more important role in the pathogenesis of PGIA. Our study shows that CFA induces more antigen-specific Th17 cells without affecting disease severity. The results from both of these studies indicate that the cytokine profile of the T cells does not have much impact on the clinical course of PGIA per se. However, whether the composition of the inflammatory joint infiltrate is similar in Th1-induced and Th17-induced arthritis remains to be addressed. In this respect it is of interest to note that studies in the experimental autoimmune encephalomyelitis model have shown that the type of effector T cell does determine disease pathogenesis. Adoptive transfer of Th1, Th9 or Th17 cells can all induce experimental autoimmune encephalomyelitis with similar clinical symptoms, but different immunopathogenesis.19 In a different study it was shown that transfer of IL-12-conditioned T cells induced more infiltration of macrophages, whereas transfer of IL-23-conditioned T cells caused a more granulocyte-rich infiltrate.20 Here, we show a difference in the Th1/Th17 balance after immunization with the different adjuvants. It is possible that this will also result in a difference in the composition of the inflammatory joint infiltrate. This issue will have to be addressed in future studies.

The fact that the adjuvant influences the T-cell cytokine profile in PGIA can also be used to make the model more versatile. PGIA can be used to either study Th17 cells after disease induction with CFA or to study Th1 cells after disease induction with DDA, making this model a useful tool for studying joint antigen-specific Th1 and Th17 responses.

The pro-Th17 polarizing effect of CFA is likely to be the result of the presence of heat-killed M. tuberculosis in this adjuvant. It has been shown that mycobacteria promote Th17 responses in vitro through the induction of transforming growth factor-β in DC21 and treatment of diabetic mice with mycobacterial adjuvant (e.g. CFA) induces IL-17 production by CD4+ T cells in vivo.22

Dimethyldioctadecyl-ammonium bromide is a potent inducer of IFN-γ production.14,23 The DDA forms liposomes and adsorption of antigen onto these DDA liposomes enhances uptake by antigen-presenting cells, increasing the efficiency of antigen presentation without enhancing the expression of CD40, CD80 and CD86.24 It has been suggested that additional stimulation of DC by CD40 ligand on T cells might be required for further DC maturation; however, there is no experimental evidence to support this hypothesis.24 The effect of DDA on cytokine production by antigen-presenting cells is as yet unknown and needs further investigation.

Another factor that impacts on T-cell polarization is the strain of mouse. It is well-established that the T-cell cytokine profile differs between different strains of inbred mice. Recently, it has been described that T cells from DBA/1 mice are more easily skewed towards a Th17 phenotype compared with T cells from BALB/c or C57BL/6 mice.25 Unfortunately, it is not possible to study whether the difference in mouse strain is responsible for the difference observed between the two models, because C57BL/6 and DBA/1 mice, the mouse strains susceptible to CIA, are not susceptible to PGIA, and BALB/c mice do not develop CIA.26,27 However, as differences in the Th1/Th17 ratio are more striking when comparing PGIA-immunized BALB/c and CIA-immunized DBA1 mice (Fig. 1) than when comparing BALB/c mice immunized with either CFA or DDA as the adjuvant (Fig. 2b), it cannot be ruled out that not only the type of adjuvant but also the mouse strain contributes to the observed differences in Th1/Th17 polarization.

Interestingly, the total level of IL-17 that was produced after antigen-specific or polyclonal stimulation was not significantly higher in CFA-immunized mice, although they did have a higher proportion of Vβ4+ Th17 cells after both polyclonal and antigen-specific stimulation. This indicates that although there are more Th17 cells, the total level of IL-17 production after antigen-specific stimulation is not affected by the adjuvant. The CFA contains heat-killed M. tuberculosis, and it has been shown that challenging mice with M. tuberculosis induces IL-17 and IFN-γ production by CD4+ T cells.28 The PPD-specific IL-17 and IFN-γ production demonstrates that PPD-specific T cells are induced after injection with CFA. It is not known whether these cells contribute to disease pathogenesis. However, the presence of these M. tuberculosis-specific T cells suggests that polyclonal stimulation of T cells to assess cytokine production may not be an appropriate method to study T-cell responses in mice immunized with CFA.

In conclusion, we show that both CFA and DDA can be used as adjuvants to induce PGIA. This choice will affect the proportion of cytokine-producing cells. Hence, PGIA can be used to either study Th17 cells (after disease induction with CFA) or it can be used to study Th1 cells (after disease induction with DDA), making this a very versatile model and a useful tool for studying joint antigen-specific Th1 and Th17 responses.

Acknowledgments

This work was funded by the JGW Patterson Foundation.

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