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MHC class II alleles associated with Th1 rather than Th17 type immunity drive the onset of early arthritis in a rat model of rheumatoid arthritis

Polymorphisms in the MHC class II (MHCII) genes are strongly associated with rheumatoid arthritis, supporting the importance of autoreactive T helper (Th) cells for the development of this disease. Here, we used pristane‐induced arthritis (PIA), induced by the non‐antigenic hydrocarbon pristane, to study the impact of different MHCII alleles on T‐cell activation and differentiation. In MHCII‐congenic rats with disease‐promoting MHCII alleles, pristane primarily induced activation of Th1 cells, whereas activated T cells were Th17 biased in rats with protective MHCII alleles. Neutralization of IFN‐γ during T‐cell activation abrogated the development of disease, suggesting that Th1 immunity is important for disease induction. Neutralization of IL‐17, by contrast, suppressed arthritis only when performed in rats with established disease. Adoptive T‐cell transfers showed that T cells acquired arthritogenic capacity earlier in strains with a prevailing Th1 response. Moreover, upon pristane injection, these strains exhibited more Ag‐primed OX40+ and proliferating T cells of polyclonal origin. These data show that T cells are polarized upon the first encounter with peptide‐MHCII complexes in an allele‐dependent fashion. In PIA, the polyclonal expansion of autoreactive Th1 cells was necessary for the onset of arthritis, while IL‐17 mediated immunity contributed to the progression to chronic disease.


Introduction
The MHC is located within a 3.6 Mb region on human chromosome 6 [1,2]. The region encodes over 250 genes of which MHCII loci, which contain genes encoding for the MHC class I and II Ag-presenting molecules. The MHCII locus also contains a number of mostly non-polymorphic genes encoding proteins involved in the processing, editing and transportation of Ags for presentation on MHC molecules [11][12][13][14].
Besides pathogen-derived Ags, MHCII molecules present self-Ags to CD4+ T cells, which is important for maintaining the size of the peripheral T-cell pool [15][16][17]. Tolerance to these self-Ags is established through elimination of autoreactive T cells in the thymus, generation of T regulatory cells (Treg cells), as well as by several peripheral mechanisms of which anergy and deletion of autoreactive cells are the best characterized [18,19]. Nevertheless, in ß3-5% of humans [20,21], these control mechanisms are not sufficient and autoimmunity ensues.
The genetic risk associated with autoimmunity is largely governed by genes in the MHC [4,22], in particular HLA-DRB1, which accounts for approximately 36% of the heritability in rheumatoid arthritis (RA) [23,24]. To study the mechanisms underlying autoimmunity, animal models have been established in which self-tolerance is broken by an active immunization with an Ag. In mice and rats, immunization with tissue-specific Ags or even ubiquitously expressed proteins induces organ-specific autoimmunity [25][26][27][28]. Such induced type of autoimmunity is often dependent on T cells recognizing a specific peptide-MHCII complex; for example, in collagen-induced arthritis (CIA), a peptide derived from type II collagen presented by H2-A q is crucial for the development of disease [29][30][31][32]. However, the ensuing autoimmunity in such models is not necessarily T-cell-driven [29,[33][34][35] or induced by an autologous T-cell epitope: mice immunized with rat, bovine or chicken CII, for example, respond strongly to stimulation with the heterologous CII but relatively weakly to autologous mouse CII [36,37].
A T-cell-driven arthritis model that is not dependent on the administration of exogenous Ags is pristane-induced arthritis (PIA) in the rat [15,18,38]. Pristane is a saturated, naturally occurring, hydrocarbon that is used for ascites production and the induction of lupus in mice [39][40][41]. In DA rats, a single intradermal injection of pristane induces a chronic relapsing polyarthritis that can remain active for at least 200 days [18]. The disease onset is early, highly synchronized between animals, and accompanied by elevated levels of IL-6, rheumatoid factor, and a strong acute phase response [15,18,42,43]. The induction of arthritis does not appear to be dependent on B cells or the production of antibodies, since adoptive transfer of CD4 T cells from rats injected with pristane induces a disease in naïve rats that closely mimics the manifestations in PIA [14,18,23,44].
Similar to RA, PIA is associated with multiple discrete loci in the MHC [15,44] of which two have been mapped at high resolution; a 33-kb locus in the MHCIII-region (Ltab-Ncr3), and a 0.2 Mb interval, denoted T cell selection QTL 2 (Tcs2) [12], in the MHCII-region. Analysis of Tcs2-congenic strains has demonstrated that certain MHCII alleles promote PIA whereas other alleles confer a protective effect [15]. Functional studies and analysis of coding variants between different Tcs2-congenic strains have suggested that RT1-B, the rat ortho-logue to human HLA-DQ, is responsible for the Tcs2-association [15].
Here, we used Tcs2-congenic strains to investigate how different MHCII alleles influence T-cell differentiation in rats injected with pristane. We show that pristane administration induces a Th1 biased immune response in rats with disease-promoting MHCII alleles and that IFN-γ, a hallmark Th1 cytokine, is necessary for the development of PIA. Moreover, upon pristane administration, T cells in strains with promoting MHCII alleles acquired an arthritogenic capacity earlier, were more proliferative and contained a larger fraction of Ag-experienced cells compared to strains with protecting MHCII alleles.

Arthritis-promoting MHCII alleles trigger an early expansion of pathogenic T cells
The 206 kb region that constitutes Tcs2 contains 12 genes of which RT1-DMb (HLA-DMB) and Btnl2 are located within the QTL borders ( Fig. 1A) [12]. We have previously shown that Tcs2-congenic strains, that is, DA.1HR61, DA.1UR10, and DA.1FR9, differ in their susceptibility to PIA due to coding polymorphisms in the RT1-B locus [15]. DA.1HR61, and to a lesser extent, DA.1UR10 show a later disease onset compared to non-congenic DA rats and are protected from severe PIA in the acute phase of the disease (i.e. the first ß40 days after pristane injection). DA.1FR9, by contrast, develops a more severe form of arthritis with an early onset. In the present study, we also used two new strains, DA.1HR10 and DA.1FR61, which refine the congenic intervals in DA.1HR61 and DA.1FR9, respectively (for a physical map over all strains, see Tuncel at al. [12]). The development of PIA in DA.1HR10 and DA.1FR61 was comparable to the arthritis development in the respective parental strains ( Fig. 1B and C; see also Supporting Information Fig. 1 for a comparison of PIA in DA.1HR10 versus DA.1HR61 and DA.1FR61 versus DA.1FR9).
We first asked whether the variation in disease onset between Tcs2-congenic strains and DA was due to the extent of priming the T cells required to become arthritogenic. To test the arthritogenicity of T cells after pristane administration, we performed adoptive cell transfers where T cells from draining lymph nodes (dLNs) were transferred from one donor into one MHCII syngeneic recipient (here referred to as "individual transfer"; see Methods). In DA rats, donor T cells required a minimum of 5 days of priming in vivo to transfer arthritis and 8 days to become fully arthritogenic (Fig. 1D). Similarly, in DA.1FR61, T cells from eight out of nine donors (89%) transferred arthritis after 5 days of priming in vivo. In contrast, this 5-day interval was only sufficient to prime T cells in 2/8 DA.1HR10 donors (25%, p<0.002 versus DA) and 6/10 DA.1UR10 (60%, p<0.2 versus DA) donors to transfer arthritis (Fig. 1E).
To identify whether the arthritogenic capacity of donor T cells was due to qualitative or quantitative differences between the Tcs2-congenic strains and DA, we first assessed the frequency of   proliferating (Ki67 + ) memory/activated T conventional (Tconv) cells (CD45RC lo CD90 -Foxp3 -CD4+ [45]) in pristane-injected rats. The frequency of proliferating cells in this subset in DA and DA.1FR61 increased by ß4-fold from day 4 to day 5 and remained high until at least day 8 (Fig. 1F). DA.1UR10 showed fewer proliferating memory/activated Tconv cells than DA and DA.1FR61 at day 5 but not at day 8, while the proliferation of these cells in DA.1HR10 was significantly lower at both time-points. This variation in T-cell proliferation was not specific for memory/activated Tconv cells but was also seen among CD8 T cells (Fig. 1F) and in other CD4 subsets such as Treg cells and CD90 + recent thymic emigrants (RTEs) in DA.1HR10 (Supporting Information Fig. 2).
Taken together, the early onset in DA and DA.1FR61 compared to DA.1UR10 and DA.1HR10 correlated with a more rapid expansion of arthritogenic effector T cells in the pristane-dLNs.

Pristane induces a polyclonal expansion of Tconv cells
The rate of T-cell proliferation was increased in strains with an early arthritis onset. To assess the clonality of proliferating Tconv cells, we analyzed T cells in the dLN from pristane-injected rats using available anti-Vβ antibodies by flow cytometry. The   Figure 2. The TCR Vβ repertoire on CD4 T cells is largely unaffected by pristane administration. CD4+ T cells were analyzed for the relative frequency of Vβ10, Vβ16.1, Vβ8.5, and Vβ8.2/8.4+ subsets in dLNs before (day 0) and 5 days after pristane injection. Stacked bars show Vβ frequencies among total CD4+ T cells (no significant differences were observed between Foxp3+ and Foxp3-CD4 T cells). Numbers within bars represent mean frequencies. Data are representative of at least three independent experiments. n = 6 rats/ group; *p<0.01 compared to DA day 0, † p<0.01 compared to same strain day 0 (Mann-Whitney U).
frequency of Vβ10, Vβ16.1, Vβ8.5, and Vβ8.2/8.4 subsets, which together constituted ß25% of the total Vβ repertoire, varied significantly between the strains (Fig. 2). Vβ10 + Tconv cells, for example, which have been shown to adoptively transfer PIA [23], were more abundant in DA and DA.1FR9 rats, while the frequency of Vβ16.1 + Tconv cells was highest in DA.1HR61 and DA.1UR10. However, except for Vβ8.5, which was more frequent in DA and DA.1FR9 after pristane administration, the relative frequency of each Vβ subset among total Tconv cells in dLNs was not affected by pristane (Fig. 2). Thus, this indicates that T cells of a broad clonal origin, of which some are likely to be autoreactive, expand in pristane-dLNs.

Antigen-experienced T cells are enriched in the dLNs of rats with arthritis-promoting MHCII alleles
Flow cytometric analyses of Treg cells in naïve rats did not reveal any differences in frequencies or numbers between susceptible and protected strains (Supporting Information Fig. 3). After pristane injection, DA.1HR10 rats showed a modest increase in the frequency of Treg cells whereas there was a reduction of Treg cells in DA.1UR10 compared to DA and DA.1FR61 (Fig. 3A), indicating that the protective MHCII alleles in DA.1HR10 and DA.1UR10 did not simply favor a selection or expansion of Treg cells over Tconv cells. However, the proportion of Treg cells that were negative for the tumor necrosis factor receptor OX40 was almost 2-fold higher in DA.1HR10 compared to all other strains (Fig. 3B, 3C). This may suggest that a significantly larger proportion of the Treg cells in DA.1HR10 are functionally suppressive since ligation of OX40 on Treg cells has been shown to turn off their suppressive capacity [29,34].
On Tconv cells, OX40 is expressed on effector cells that are Ag-primed or have undergone TCR ligation [25,27,46]. CD4 T cells in dLNs from DA and DA.1FR61 showed significantly higher proportions of OX40+ cells on day 5 and day 8 after pristane administration compared to DA.1HR10 ( Fig. 3B and 3D). This early expansion of Ag-primed T cells in DA and DA.1FR61 likely explains why T cells in these strains were more arthritogenic on day 5 than T cells in DA.1HR10 and DA.1UR10 (Fig. 1E). Moreover, when the variation in Treg cell frequency was considered ( Fig. 3A), the Treg/T effector cell ratio was approximately twofold higher in DA.1HR10 compared to DA and DA.1FR61 on day 5 and 8 after pristane administration (Fig. 3E). Together, these data suggest that promoting MHCII alleles accelerate the onset of PIA by expanding Ag-specific T cell clones in the pristane dLNs. 24

D C E A B
Days after pristane injection

Activated T cells are predominantly of Th1 type in rats with disease-promoting MHCII alleles
To assess differences in T-cell responses between the strains, we determined mRNA levels of cytokines and Th-lineage-defining transcription factors in purified CD4 T cells from pristane-dLNs. This showed that IFN-γ was upregulated in strains with promoting MHCII alleles and, surprisingly, a distinct upregulation of Th17 signature genes (IL-17A, IL-22, and RORγt) in T cells from DA.1HR10 (Fig. 4A). A similar shift in Th1/Th17 associated cytokines was observed when stimulating T cells from pristaneimmunized DA and DA.1HR10 rats with anti-CD3/CD28 in vitro (Fig. 4B).
Flow cytometric analyses of CD4 T cells showed that IFN-γ producing Th1 cells increased by approximately twofold between day 3 and day 5 in DA and DA.1FR61 rats (Fig. 4C). By contrast, the frequency of Th1 cells in DA.1HR10 and DA.1UR10 was only marginally increased during the first 5 days of PIA. Further, the total number of Th1 cells was significantly increased in dLNs from DA and DA.1FR61 compared to DA.1HR10 and DA.1UR10 on day 5 and 8 after immunization. (Fig. 4D). Unlike unstimulated cells (Fig. 4A) and cells stimulated with anti-CD3/CD28 (Fig. 4B), PMA/ionomycin stimulation did not reveal any significant differences in IL-17 expression between the strains (data shown for total numbers of Th17 cells in Fig. 4D).
Taken together, these data show that after pristane administration T cells differentiate predominantly into Th1 lineage in rats with arthritis susceptible MHCII alleles.

Pristane-induced arthritis is dependent on IFN-γ for T-cell priming and IL-17 for disease perpetuation
A significant expansion of Th1 cells was observed in strains with an early-onset PIA. To probe whether IFN-γ is critical for the induction of arthritis, we treated rats with anti-IFN-γ and anti-IL-17 neutralizing antibodies at day 2, 4, and 6 after pristane administration.
All rats, regardless of MHCII haplotype, that were treated with anti-IFN-γ Abs developed significantly milder disease compared to non-treated control rats (Fig. 5A), whereas no effect was observed when treating with anti-IL-17 Abs (Fig. 5B). In addition, the onset of PIA was delayed by up to 4 days in rats treated with anti-IFNγ Abs (Fig. 5C), suggesting that T-cell priming in PIA is IFN-γdependent also in strains associated with a Th17 biased immune response, such as DA.1HR10.
We next assessed whether IFN-γ and/or IL-17 was required for the progression of established arthritis by injecting neutralizing antibodies at day 8, 10, 12, and 14 after pristane administration (Fig. 6). The effect of IFN-γ neutralization on arthritis progression was negligible or promoting ( Fig. 6A-6C) in all strains except in DA.1UR10 where it slowed disease development (Fig. 6D). Neutralization of IL-17, by contrast, was highly efficient in suppressing the progression of disease in all strains (Fig. 6A-D). This effect of IL-17 neutralization was followed by a twofold reduction in circulating neutrophils (Fig. 6E), suggesting that IL-17 operates on granulopoiesis and the recruitment of neutrophils to the joints in PIA.

FR9
Mean arthritis score

Discussion
There is compelling evidence that IL-17 contributes to the pathology in a number of autoimmune diseases [47,48]. IL-17 (or IL-23) deficient mice, for example, are protected from arthritis and depletion of IL-17 has been shown to suppress both priming of T-cell responses as well as the progression of ongoing disease [49][50][51][52].
In addition, IL-17 is enriched in the blood and synovial fluid of patients with RA, suggesting that IL-17 might be important also in human RA [53][54][55]. In comparison, the role of IFN-γ in autoimmune arthritis has been more controversial, probably as its modus operandi can be both pro-inflammatory and anti-inflammatory depending on context [56]. Here, we show that activation of autoreactive T cells through administration with a non-antigenic hydrocarbon skews the immune response toward Th1 and that neutralization of IFN-γ protects rats from developing severe arthritis. Importantly, we found that an early Th1 signature dominated in rats expressing arthritis-promoting MHCII alleles, providing a link between effector T-cell differentiation, disease phenotype and MHCII genotype. The MHCII locus confers the highest genetic risk in RA [57] and the major impact of permissive MHCII alleles is likely to be on the early disease development. Although poorly characterized in humans, the preclinical stage of arthritis is likely to involve a series of linked events that eventually cause a breech in tolerance, activation of T and B cell responses, and an attack on peripheral joints. However, these events do not necessary need to unfold in the joints. In fact, telomeric length studies have shown that T cells are recruited to the joints in a preactivated state [1] suggesting that the joint is not the primary organ for a cognate T-cell response. It has also been suggested that joint-infiltrating T cells are of polyclonal origin; however, the Ag-specificities of these T cells remain largely unknown even if post-translationally modified peptides [3], and peptides with weak affinity to MHCII [5,7] could serve as potential target Ags.
A similar series of events, albeit in a much-compressed time frame, may lead to the development of PIA in rats. First, pristane, being a hydrocarbon, cannot bind to MHCII molecules and it is unlikely that pristane operates directly on T cells or is presented by CD1 [9]. Instead, functional and genetic data have suggested that stimulation of innate signals and pattern-recognition receptors (PRRs), such as Toll-like receptors and C-type lectin receptors, are important for the triggering of an adaptive response in PIA [11,13,14]. Although it has never formally been excluded that pristane cannot be presented by CD1, blocking of MHCII-TCR interactions during T-cell priming (2-4 days post pristane) is sufficient to ameliorate PIA [15]. Second, activation of skin-resident dendritic cells appears to be important for the priming of autoreactive T cells in PIA [18]. Third, injections of 14C-labelled hexadecane have shown that mineral oils accumulate within the dLNs with no detectable oil being found in the joints themselves [20], suggesting that the primary site of T-cell activation is within the lymphoid organs. Our data suggest that the primary sites of T-cell activation are LNs draining the injection site as T cells at these locations could transfer arthritis as early as 5 days after pristane administration. We show that expanded T cells in dLNs are of broad clonality, which is consistent with previous studies showing that arthritogenic T cells in PIA use a diverse repertoire of TCR Vβ chains [23]. The co-stimulatory molecule OX40 is specifically upregulated on Ag-primed T cells [25,27] and has been shown to promote clonal expansion and survival of T effector and memory cells [29,31]. We assessed early OX40 expression in pristane dLNs and found that OX40 positive T effector cells (CD45RC lo CD90 -Foxp3 -) were enriched in the dLNs of rats carrying arthritispromoting MHCII alleles. OX40 expression peaked around day 5 after pristane injection, which coincides with the emergence of arthritogenic T cells in DA and DA.1FR61 rats and the establishment of a Th1 signature in these strains. Together, these findings provided a possible explanation for the early arthritis onset associated with certain MHCII alleles; namely, the rapid expansion and differentiation of Ag-primed T cells to arthritogenic effector cells.
In contrast to its role on Tconv cells, the role of OX40 on Treg cells remains controversial. Several studies have shown that ligation of OX40 on Treg cells dampens their regulatory activity [29,34], and, in the presence of IFN-γ and IL-4, favors expansion of T effector cells over Treg cells [36]. This could explain the expansion of T effector cells we observed in DA and DA.1FR61 after pristane administration, where the local cytokine milieu, being high in IFN-γ, would favor T effector over Treg cell proliferation. Moreover, the high expression of OX40 on Treg cells in DA and DA.1FR61 could be associated with a poor immune regulatory function in these strains, which could amplify an ongoing expansion of autoreactive T cells even further. Consistent with this notion, also CD8 T cells, which are not restricted by MHCII or essential for the development of PIA [15], showed a greater proliferation in strains with arthritis promoting MHCII alleles. Such "by-stander" proliferation could be associated with OX40 expression on Treg cells, since IL-2 and other growth factors could become more available in the absence of sufficient Treg suppression. Further, this could mean that Treg cells in DA.1HR10, which consisted of a larger proportion of OX40 negative cells, are better suppressor cells than Treg cells in DA and DA.1FR61. However, OX40 deficiency in Treg cells has also been associated with a lack of immune suppression, as shown for T-cell-induced colitis [39]. Hence, what implications the differential expression of OX40 may have on Treg cells in PIA and what relevance this may have for the protection observed in DA.1HR10 remains to be determined.
Effector T cells in rats carrying disease promoting MHCII alleles differentiated to Th1 cells upon pristane administration. This Th1 signature is likely to be responsible for the early onset and severe progression of arthritis in DA and DA.1FR61 since neutralization of IFN-γ in pre-arthritic rats delayed the onset and reduced the severity of PIA. Hence, depleting IFN-γ during T-cell priming in DA and DA.1FR61 rats seems to phenocopy the characteristic "DA.1HR10 disease". Unlike DA and DA.1FR61, DA.1HR10 rats did not demonstrate a propensity for developing Th1 immunity. Instead, CD4 T cells from DA.1HR10 expressed higher levels of IL-17, IL-22 and ROR-γt compared to DA and DA.1FR61, suggesting that pristane drives the expansion of Th17 cells in DA.1HR10. Importantly, this expansion of IL-17 producing T cells did not seem to be pathogenic since depletion of IL-17 in pre-arthritic rats did not influence the onset or progression of PIA in DA.1HR10 or in any of the other strains. Interestingly, the frequency and total number of IFN-γ producing Th1 cells increased over time in both DA.1HR10 and DA.1UR10, and reached the levels of DA and DA.1FR61 by the time of arthritis onset. This suggests that it is the expansion of autoreactive Th1 cells that ultimately determines the onset of PIA.
Th1 type immune responses have previously been demonstrated to be important for the induction of G6PI-induced arthritis [58] and neutralization of IFN-γ inhibits arthritis in proteoglycaninduced arthritis (PGIA) [59,60]. Recently, the formation of neutrophil extracellular traps (NETs) was shown to induce expansion of Th1 cells and accelerate the onset of CIA in mice [61]. Interestingly, pristane has been shown to be a potent inducer of NETs [62], suggesting that NETs could serve as an inducer of Th1 immunity in PIA. Th1 mediated immunity may also be relevant in early RA; studies have demonstrated that the preclinical phase of RA is dominated by Th1-associated cytokines and chemokines (IFN-γ, IL-12, and CXCL10) [63,64]. Moreover, lymph node biopsies taken from individuals with an increased RA-risk (that is, IgM-RF and/or ACPA positive individuals with arthralgia but without any evidence of arthritis) and from patients with early RA showed elevated levels of T cells with a Th1 profile (CXCR3+ CCR6− CCR4−) as compared to healthy controls, whereas no such differences were found for T cells with a Th17 or Th2 profile [65]. In established RA, however, the presence of Th1 cells seems to be associated with disease protection rather than exacerbation. Likewise, in the PGIA model, IFN-γ deficient mice eventually succumb to severe arthritis, probably as a result of increasing IL-17 levels [59]. In PIA, DA.1HR10 rats develop the same severe chronic arthritis as non-congenic DA rats [15], which is consistent with the finding here that the onset of disease marks the transition from an IFN-γ to an IL-17 dependent disease. Whether Th17 cells are the primary source of IL-17 in established PIA remains to be determined. Unpublished findings from our lab show that both IL-17 and T cells are important for the perpetuation of late-stage chronic PIA, suggesting that Th17 cells may play a specific role in the transition to chronic disease (J.T., S.H., and R.H., unpublished results). This would be consistent with the notion that synovial infiltration of Th17 cells may play a specific role in the transition from acute synovitis to chronic inflammation in RA [49,66].
We have shown that a Th1 type immune response is essential for the development of PIA and that Th1 skewing is solely dependent on the MHCII allele. Tcs2-congenic rats carrying MHCII alleles that skewed the immune response toward Th1 had more Ag-primed T cells in the dLNs, developed a more severe arthritis and showed an earlier disease onset than rats carrying MHCII alleles that skewed the immune response toward Th17. While a Th1-type immune response was essential for the onset of PIA, IL-17 mediated immunity was necessary for the establishment of chronic arthritis.

Materials and methods
Animals DA/OlaHsd (Harlan Europe, The Netherlands) were maintained in a barrier facility by sister-brother mating and were specific pathogen free (SPF) according to the current FELASA guidelines [67], which includes pathogens such as Parvovirus, Sendai virus, Hantaan virus, Coronavirus, Reovirus, Cytomegalovirus, Pasteurellaceae and Mycoplasma pulmonis. Animals were kept in a climate-controlled environment with 14 h light/ 10 h dark cycles, housed in individually ventilated microisolator-cages (IVC) containing wood shavings and fed standard rodent chow and filtered water ad libitum. The derivation of congenic strains has been described previously [12]. All rat strains are available as an open resource at the Rat Resource and Research Center, Columbia, MO.

PIA induction
The induction and evaluation of PIA in DA rats have been described recently [18]. In brief, PIA was induced by a single intradermal injection of 100 μL pristane (2,6,10,14-tetramethylpentadecane, 95%, Acros Organics, Morris Plains, NJ, USA) at the dorsal side of the tail base. All experiments were performed in 8-11 weekold, sex and age-matched (±5 days) rats. Disease protected and non-protected strains (or different treatment groups) were housed together in cages from the time-point of weaning or for at least 2 weeks before the start of the experiment. Arthritis development was monitored daily or every second day using a macroscopic scoring system of 1-60 points per rat as described previously [18]. In brief, 1 point was given for each inflamed knuckle or toe and up to 5 points were given for an affected ankle (in total 15 points per paw). All scoring was performed blinded and by different investigators. Weight loss of pristane-injected rats was included as an objective measure of the disease severity. Weight changes in figures are indicated as % of body weight at disease onset. All experiments were approved and performed in accordance with the guidelines from the Swedish National Board for Laboratory Animals and the European Community Council Directive (86/609/EEC).

Individual transfer
Adoptive PIA transfer has been described previously [23] but was modified here as follows: A single cell suspension from a pair of inguinal LNs was prepared at RT in 5 ml transfer medium containing 3 μg/mL Concanavalin A (Con A, Sigma Aldrich, C5275), 5% FCS (Gibco), 2.4 mg/mL HEPES, 3.9 μg/mL 2-ME, 10 4 IU/mL penicillin, and 10 mg/mL streptomycin (both Invitrogen Life Technologies) in Dulbecco's MEM (Gibco), using a 40 μm cell strainer (BD Falcon). The cell strainer was rinsed with 3 mL of transfer medium, and 1.5 × 10 8 cells were added to a 75 cm 2 culture flask (2 × 10 6 /cm 2 ). Transfer medium was added to a final volume of 50 mL and the cells were thereafter incubated for 70 h in a humidified 5% CO 2 atmosphere at 37°C. Con A-stimulated cells were transferred to centrifugation tubes (BD Falcon), pelleted at 300 x g for 12 min at RT, and washed once in sterile PBS. The pelleted cells were gently resuspended (without adding new PBS) and then transferred to a 40 μm cell strainer placed over a clean 50 mL centrifugation tube. After washing the original tube with 200 μL of PBS, the tube was centrifuged (with the cell strainer attached) for 10 s at 200 x g to collect cells. The cell concentration was adjusted with PBS to 10 8 cells/mL. Naive (non-irradiated) MHC-II syngenic recipients were injected intravenously (i.v.) with 4 × 10 7 cells.

Flow cytometry
Cells were washed in cold EDTA-FACS buffer containing 1% bovine serum albumin [BSA, Sigma-Aldrich], 2 mM Na 2 EDTA [Merck, Darmstadt, Germany] and 0,02% NaN 3 in PBS-D and resuspended in the same buffer. For staining with MAbs, 10 6 cells/well were added in duplicates to 96-well v-bottom polypropylene plates (BD Falcon). For cytokine staining, cells were resuspended in transfer medium (see above) without Con A and seeded at 10 6 cells/well (100 μL) in duplicates in tissue-culture treated 96-well u-bottom plates. The cells were mixed with an equal volume of the same medium containing 10 ng/mL Phorbol 12-Myristate 13-Acetate (PMA), 0.6 μg/mL ionomycin and 10 μg/mL Brefeldin A (all from Sigma-Aldrich) and incubated for 4 h at 37°C in a 5% CO 2 atmosphere. PMA-stimulated cells were transferred to v-bottom plates, washed once in cold EDTA-FACS buffer, stained extracellularly with MAbs, fixed and permeabilized in BD Cytofix/Cytoperm for 20 min at RT followed by intracellular staining. Cells cultured in the absence of PMA and ionomycin served as negative controls. For the intracellular detection of Ki67 and Foxp3, cells were incubated for 45 min at RT in Fixation/Permeabilization buffer (eBioscience) and thereafter washed twice in a saponin-containing buffer (BD Perm/Wash) before staining with anti-Foxp3 and anti-Ki67. Fluorescence-minus-one (FMO) controls were used in all experiments. LIVE/Dead Violet (Invitrogen, Carlsbad, CA) was used to gate live cells during analyses. Duplicate samples were pooled prior to acquisition on a BD LSR-II Flow Cytometer. All data were analyzed with FlowJo (Tree Star Inc., Ashland, OR).

Treatment with anti-IFN-γ and anti-IL-17
DB-1 (anti-INF-γ, mouse IgG1) was produced from B cellhybridoma (kindly provided by Dr. Tomas Olsson) as described previously [15]. Anti-mouse IL-17A antibody (clone 17F3; mouse IgG1) and isotype control antibody (clone MOPC-21) was obtained from Bio X Cell (West Lebanon, NH, USA). Crossreactivity to rat IL-17 was evaluated by ELISA using supernatant from Con A-activated cell cultures. Antibodies were administrated s.c. in 0.1 mL sterile PBS (1 mg/mL) at each flank of the tail base (0.2 mL/ 0.2 mg in total per rat and time point) as described previously [18] to target the pristane dLNs during the priming phase of PIA (day 2, 4, 6 after pristane injection). Antibodies for treatment of early-established disease (day 8, 10, 12 and 14 after pristane injection) were administrated i.v. using the same volumes and concentrations as used for s.c. injections.

RNA extraction and qRT-PCR
Cell pellets were allowed to thaw slightly before being resuspended in 300 μL RLT buffer (QIAGEN Nordic, Ballerup, Denmark) containing 10 μL/mL 2-ME. Automated RNA isolation was performed on a QIACube robot using RNeasy extraction reagents (Qiagen) with on-column DNase I digestion (Qiagen). RNA quantity and purity was assessed on a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE). RNA samples were diluted to 10 ng/μL in DEPC treated water (Ambion). Complementary DNA (cDNA) was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in 50 μL reactions. Primers were designed in Primer-BLAST (ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) or obtained from RTPrimerDB (medgen.ugent.be/rtprimerdb) (Table S1). SYBR-Green PCR master mix (Applied Biosystems, Foster City, CA) was used for all PCR reactions according to the manufacture's recommendation. Quantitative RT-PCR was performed on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Specificity and efficiency of all primers were validated prior to use by absolute quantification. The expression fold-change of each target gene was determined by the relative quantification method ( Ct) after normalization to the geometric mean of the reference genes Arbp, β-Actin and GusB.