Exacerbation of Collagen-Induced Arthritis by Oligoclonal, IL-17-Producing γδ T Cells

Christina L. Roark, Jena D. French, Molly A. Taylor, Alison M. Bendele, Willi K. Born and Rebecca L. O’Brien
J Immunol October 15, 2007, 179 (8) 5576-5583; DOI: https://doi.org/10.4049/jimmunol.179.8.5576

Abstract

Murine γδ T cell subsets, defined by their Vγ chain usage, have been shown in various disease models to have distinct functional roles. In this study, we examined the responses of the two main peripheral γδ T cell subsets, Vγ1+ and Vγ4+ cells, during collagen-induced arthritis (CIA), a mouse model that shares many hallmarks with human rheumatoid arthritis. We found that whereas both subsets increased in number, only the Vγ4+ cells became activated. Surprisingly, these Vγ4+ cells appeared to be Ag selected, based on preferential Vγ4/Vδ4 pairing and very limited TCR junctions. Furthermore, in both the draining lymph node and the joints, the vast majority of the Vγ4/Vδ4+ cells produced IL-17, a cytokine that appears to be key in the development of CIA. In fact, the number of IL-17-producing Vγ4+ γδ T cells in the draining lymph nodes was found to be equivalent to the number of CD4+αβ+ Th-17 cells. When mice were depleted of Vγ4+ cells, clinical disease scores were significantly reduced and the incidence of disease was lowered. A decrease in total IgG and IgG2a anti-collagen Abs was also seen. These results suggest that Vγ4/Vδ4+ γδ T cells exacerbate CIA through their production of IL-17.

Collagen-induced arthritis (CIA)3 is a murine model of chronic inflammation that shares many hallmarks with rheumatoid arthritis (RA) (reviewed in Ref. 1). For example, there is a strong association with the MHC class II allele HLA-DR4 (DRB1*0401) in humans and IAq in mice (2, 3) and both class II molecules bind the same immunodominant collagen type II (CII) peptide (4). In addition, autoantibodies to type II collagen have been found in the sera and synovial fluid of RA patients (5, 6, 7) and play a critical role in the development of CIA in mice (reviewed in Ref. 1). Finally, αβ T cells have been shown to be essential in CIA (8).

There is also evidence that γδ T cells play a role in CIA (9, 10). γδ T cells are resident in the synovium of mice and their proportion in the joints rises dramatically when mice develop CIA (9, 10). Similarly, γδ T cells are increased in the peripheral blood and synovium of patients with RA (11, 12, 13). However, studies in mice genetically deficient for T cells have shown that γδ T cells are neither necessary nor sufficient for the development of CIA (8). Yet, when mice were depleted of γδ T cells using a mAb, an effect on disease was noted. Depleting mice of γδ T cells before immunization with CII significantly delayed the onset of arthritis and severity. In contrast, Ab administered 40 days after the immunization resulted in rapid and severe exacerbation of CIA (9). This differential effect on the development of CIA could be explained by responses of distinct γδ T cell subsets at different time points.

Previous studies have demonstrated that the two main peripheral γδ T cell subsets (14, 15), expressing Vγ1 and Vγ4, have different functional roles in various disease models (reviewed in Ref. 16). In the CIA model, we found while both Vγ1+ and Vγ4+ cells increased, only the Vγ4+ cells were activated, as measured by surface marker expression. Because the proinflammatory cytokine, IL-17, has been shown to play an important pathogenic role in autoimmune diseases such as experimental allergic encephalomyelitis and CIA (reviewed in Ref. 17), we also examined whether γδ T cell subsets could produce IL-17. We found that the vast majority of the responding Vγ4+ cells produced IL-17 and coexpressed Vδ4. Sequence analysis revealed limited γ and δ junctional regions, indicating that these cells had undergone Ag selection. Finally, depletion of Vγ4+ cells during CIA resulted in less severe disease, indicating a pathogenic role for these cells.

Materials and Methods

Animals

Eight- to 10-wk-old DBA/1 lac J male mice (The Jackson Laboratory) were used for this study. The National Jewish Institutional Animal Care and Use Committee board approved all experimental protocols used in this work.

CIA injections

Mice were injected with bovine CII (Elastin Products) emulsified in CFA as previously described (18). Mice were scored for severity of disease every other day starting on day 21 until they were sacrificed on day 41. The following scale was used: 0, no redness or swelling; 1, one digit swollen; 2, two digits swollen; 3, three digits swollen; and 4, entire paw swollen with ankylosis. The scores for each of four paws were added together to give a final score, such that the maximal severity score was 16. In some experiments, control mice were injected with PBS emulsified in CFA. These mice did not develop disease.

Analysis of γδ T cells

We have used the simple numbering system of Heilig and Tonegawa (19) for the murine γ and δ genes. Official nomenclature equivalents are shown in parentheses (20): Vγ1 (GV5S1), Vγ4 (GV3S1), Vδ4 (DV104S1), Vδ5 (DV105S1), and Vδ6.3 (ADV7S1). On various days, the draining (inguinal, popliteal, and brachial) lymph nodes were removed for analysis. A cell suspension from the lymph nodes was made, T cells were enriched by passage over nylon wool (21) and cells were stained for γδ T cell subsets using a FITC-labeled pan Cδ Ab (GL3 (22)), followed by biotinylated anti-Vγ1 (2.11 (15)), or anti-Vγ4 (UC3-10A6 (23)) Abs plus streptavidin-allophycocyanin, and PE-conjugated anti-CD62L, anti-CD45RB, or anti-CD44 Abs (BD Biosciences). All samples were analyzed on a FACSCalibur or FACScan flow cytometer (BD Biosciences), and the data were processed using FlowJo 6.4.1 software (Tree Star).

Treatment with depleting Abs

Mice were injected with 200 μg of either an anti-Vγ4 Ab (UC3), an anti-Vγ1 Ab (2.11), or with hamster IgG, as a control, on day 17, 4 days before the booster immunization with CII/CFA. On day 21, heparinized blood samples were incubated in Gey’s solution for 10 min to lyse the RBC. Nylon wool nonadherent cells were stained with anti-CD3 (KT3 (24)), an anti-Cδ Ab (GL3), and either anti-Vγ1 (2.11) or anti-Vγ4 (UC3) Abs to verify the depletion of the appropriate subset.

Measurement of anti-collagen Abs

Serum was obtained by aspiration of retro-orbital blood on days 0 and 21, at the time of the first and the second CII injection. On day 41, mice were tail bled before being sacrificed. Each serum sample was analyzed for total IgG, IgG1, and IgG2a Ab levels to CII using modifications of published ELISA methods (18). All serum samples were diluted 1/9000 in PBS before analysis by ELISA. The plates were developed for 5 min by adding 50 μl of 3,3′,5,5′-tetramethylbenzidine substrate before the reaction was stopped with the addition of 25 μl of 2 N H2SO4. Absorbance was measured at 450 nm on a VERSAmax microplate reader and the data were analyzed using Softmax Pro 4.7.1 software (Molecular Devices). A standard pool of anti-collagen Abs was obtained by combining sera from several diseased mice and set as equivalent to 1000 U/ml.

Histology

On day 41, forepaws and hind paws (including the paw and ankle) were surgically removed and fixed in 10% buffered formalin. Tissue was prepared and histological analyses were performed as previously described (25). The treatment and clinical disease activity score of each sample was not disclosed to the trained observer who scored the slides. Sections were scored for mean inflammation, pannus formation, cartilage damage, and bone damage, and the overall score was based on a set of three to four joints per animal. All were scored on a 0–5 scale, as previously described (25). A mean score for each animal was determined for each parameter, and these were averaged to determine group means.

Isolation of cells from the joint

A modified version of a lung digestion protocol was used on the joints of mice (26). Briefly, the skin was removed to ensure that γδ T cells present in the skin were not included and then whole paws were dissected into small pieces. The pieces were placed in 0.125% dispase II (Roche), 0.2% collagenase II (Sigma-Aldrich), and 0.2% collagenase IV (Sigma-Aldrich), and shaken for 75 min at 37°C. After digestion, the supernatant was removed and the joint pieces were pushed through a Cellector tissue sieve (Bellco Glass) to disperse the cells. RBC were removed and the cell suspension was passed over nylon wool columns before staining.

Intracellular cytokine staining

Cells were cultured at 1 × 106 cells/ml in culture medium (27) containing 10 μg/ml brefeldin A (Sigma-Aldrich), 50 ng/ml PMA (Sigma-Aldrich), and 1 μg/ml ionomycin (Sigma-Aldrich) at 37°C for 4 h. After activation, cells were washed and stained with anti-CD3-allophycocyanin-AF750 (eBioscience), FITC-labeled anti-TCRδ (GL3), biotinylated or FITC-labeled anti-Vγ1 (2.11) or anti-Vγ4 (UC3), and biotinylated anti-Vδ4 (GL2 (22)), anti-Vδ5 (F45.152 (28)), or anti-Vδ6.3 (17C (29)) Abs and detected with streptavidin-allophycocyanin (BD Biosciences). The cells were then fixed in 1% paraformaldehyde for 20 min at 4°C. Fixed cells were permeabilized for 10 min at 4°C in 5% saponin/PBS buffer. Cells were stained with PE-conjugated anti-cytokine Abs (IL-2, IL-17, IFN-γ, and TNF-α; BD Biosciences) or an isotype control for 30 min at 4°C. Cells were washed once in saponin buffer and once in staining buffer before fixation.

Statistical analyses

All statistical analyses were performed using GraphPad Prism version 4 (GraphPad Software). Statistical significance for the clinical disease activity was determined using the Mann-Whitney U test. For incidence of disease, the log-rank test was used to determine significance. The histological data were analyzed by comparing group means using the Student t test with significance set at 5%. For the anti-collagen Abs, statistical significance was determined using an unpaired two-tailed Student t test to compare the two treatment groups.

Sequencing of TCRs

Total cell RNA was isolated from nylon wool nonadherent cells obtained from the lymph nodes of mice using the PicoPure RNA Isolation kit (Arcturus Bioscience). RT-PCR was performed using primers specific for Vγ4/Cγ1, 2, and Vδ4/Cδ as previously described (27). PCR-amplified transcripts were cloned into a TA vector (Invitrogen Life Technologies) and individual clones were sequenced to determine the frequency of specific TCR sequences.

Results

γδ Τ cell subsets respond differentially in CIA

To further define the role of γδ T cells in CIA, we analyzed the two main lymphoid γδ T cell subsets in mice on various days after collagen/CFA injection. Nine days after the first injection, total γδ T cells were increased ∼3-fold when compared with untreated mice (day 0) (Fig. 1⇓A). Within 3–4 days following the second immunization, total γδ T cells increased again (Fig. 1⇓A). The responses of both the Vγ1+ and Vγ4+ γδ T cells mirrored that of total γδ T cells, and both increased in numbers to approximately the same degree after the first collagen/CFA injection. However, Vγ4+ cells increased rapidly after the second injection (right panel, Fig. 1⇓B), while Vγ1+ cells increased more slowly and less vigorously (left panel, Fig. 1⇓B).

FIGURE 1.
FIGURE 1.

The total numbers of γδ T cells, Vγ1+ cells, and Vγ4+ cells obtained from mice that had received collagen/CFA injections on days 0 and 21 (black arrows). On various days, the draining lymph nodes (inguinal, brachial, and popliteal) were removed and cells were stained for γδ T cell subsets. Using FACS analysis, the total number of γδ cells (A) and individual subsets (B) were calculated. Each time point represents the average + SEM for at least eight different mice. C, On designated days after collagen/CFA injections (black arrows), γδ T cells were stained for Vγ1 and Vγ4 expression and for levels of CD62L, CD44, or CD45RB. The mean percentage + SEM of cells having an “activated” phenotype (CD62Llow, CD44high, CD45RBlow) is shown.

The loss of CD62L and CD45RB expression along with the gain of CD44 have been shown to correlate with αβ T cell activation/memory (30). Therefore, we also stained the γδ T cell subsets for these markers at various time points after CII immunization. As shown in Fig. 1⇑C, the percentage of Vγ4+ cells (right panel) that expressed high levels of CD44 increased (>10-fold) within the first 9 days of the disease course. A reciprocal loss of CD62L and CD45RB expression was also seen. These “activated” cells were transient and returned to near baseline levels during the first 3 wk of the disease process. Following the second immunization, the percent of activated Vγ4+ cells again increased. In contrast, Vγ1+ cells (left panel) exhibited little change in expression of CD44, CD45RB, and CD62L, even though Vγ1+ cell numbers increased during CIA (Fig. 1⇑B). Therefore, the Vγ4+ subset appeared to be specifically responsive to the immunizations, whereas the Vγ1+ subset did not.

Vγ4+ γδ T cells produce IL-17 in the draining lymph nodes and joints

We next analyzed the cytokine potential of each γδ T cell subset. Draining lymph nodes were harvested on day 26, when the total number of Vγ4+ cells reaches its peak, and intracellular cytokine staining was used to detect IFN-γ, IL-2, TNF-α, and IL-17 production. In naive mice, 6% of total γδ T cells, <1% of Vγ1+ cells, and 20% of Vγ4+ cells produced IL-17 (data not shown). However, in CIA mice, 40% of γδ T cells produced IL-17 (Fig. 2⇓A). When the γδ T cell subsets were analyzed, only 1.9% of Vγ1+ cells as compared with 60% of Vγ4+ cells produced IL-17 (Fig. 2⇓A). In fact, Vγ4+ cells represented over 90% of the total γδ T cells that produced IL-17 in CIA. In contrast, the fraction (data not shown) and number of Vγ1+ and Vγ4+ cells that produced TNF-α, IL-2, and IFN-γ were similar (Fig. 2⇓B). Because IL-17 is an inflammatory cytokine produced by activated CD4+ αβ T cells (Th17 cells) (31, 32, 33, 34), we also compared the number of CD4+ cells and Vγ4+ cells that produced IL-17 in our model of CIA. Remarkably, despite its small size, the Vγ4+ population contained as many or more IL-17 producers than all CD4+ αβ T cells taken together, suggesting that Vγ4+ cells are an important source of IL-17 (Fig. 2⇓B). We also characterized the cytokine potential of γδ T cells from the joints of normal DBA/1 mice and CIA mice. Here, whole paws were digested after removing the skin. Live CD3+ cells were first gated before subsequent analysis (Fig. 3⇓A). We found a substantial percentage of TCR γδ+ cells among T cells isolated from the joints of normal animals (15%) and even more, ∼23%, in the joints of diseased paws (Fig. 3⇓B). The percentage of Vγ4+ cells was also increased among T cells from diseased joints, whereas the percentage of Vγ1+ cells was decreased when compared with those from normal joints. In addition, a large fraction (78.2%) of Vγ4+ cells taken from the joints produced IL-17 on day 26 of the disease process (Fig. 3⇓C).

FIGURE 2.
FIGURE 2.

Intracellular cytokine staining of T cells from the draining lymph nodes on day 26. A, The percentages of CD4+, γδ+, Vγ1+, or Vγ4+ T cells that can produce IL-17 are indicated, first gating on cells that stained with CD3 (top two panels). The percentage of Vγ1+ and Vγ4+ cells was visualized by next gating on cells that stained with a pan-γδ-reactive mAb (bottom two panels). B, The number of Vγ1+, Vγ4+, or CD4+ cells that produced IL-17, IFN-γ, IL-2, or TNF-α following intracellular cytokine staining, was calculated from the percentage that stained in A and B.

FIGURE 3.
FIGURE 3.

γδ T cell subsets are present in the normal joint and increased in diseased joints. DBA/1 lac J mice were injected with collagen/CFA and whole paws analyzed on day 35. Joints were stained and compared with joints taken from mice that had not received any injections. Only visibly diseased joints from collagen-injected mice were analyzed. A, Live cells were gated based on their forward and size scatter and stained for CD3 expression. B, The percentage of CD3+/γδ+ T cells, Vγ1+ and Vγ4+ γδ T cell subsets in the joints on day 26. C, Intracellular IL-17 staining of Vγ4+ γδ T cells from the joints of CIA mice on day 26 of the disease process is also shown.

CIA-elicited Vγ4+ cells preferentially express Vδ4

Although the function of mouse γδ T cells has been shown to primarily segregate with Vγ chain usage (9), a study by Shin et al. (35) implied that some γδ T cells recognize their ligand primarily through the junctional region of the δ-chain. Therefore, we looked at the δ-chains coexpressed by CIA-elicited Vγ4+ cells. Surprisingly, we found that 84% of the CIA-elicited Vγ4+ cells coexpressed Vδ4, and that these cells also represented the vast majority of the IL-17 producers (Fig. 4⇓A). In naive animals, the frequency of Vγ4+ cells coexpressing Vδ4+ was ∼20% (data not shown). Of the few Vγ4/Vδ5+ cells in the lymph nodes of the CIA mice, only a small percentage produced IL-17 (Fig. 4⇓B). Very few Vγ4/Vδ6.3+ cells were detected (data not shown). Sequence analysis of day 26 lymph node cDNA revealed a strikingly limited junctional region in the Vγ4 chain, suggesting an Ag-driven clonal response (Fig. 5⇓A). Specifically, 88% of the Vγ4+ sequences (37 of 42) encoded identical CDR3 regions that contained a leucine, encoded by N or P nucleotides, in the V-J junctional region. Multiple codon triplets were found encoding this leucine, suggesting that this population did not result from a single clonal expansion. Instead, the oligoclonal response may have been driven by specific ligand recognition. The Vδ4 sequences were also limited in variability, with most showing both length conservation (5–6 aa between V and J) and exclusive use of a single Dδ2 reading frame (Fig. 5⇓B). In addition, two conserved arginine codons were found in nearly all δ sequences, the first encoded by either the 3′ end of the Vδ4 gene or by N additions, and the second encoded by the 3′ end of the Dδ2 gene, both generated by multiple arginine codons. Small groups of identical Vδ4 clones were also evident. In contrast, Vγ4 and Vδ4 sequences from naive DBA/1 mice were highly variable (Fig. 5⇓, C and D, respectively). Importantly, no identical Vδ clones were found in the naive animals.

FIGURE 4.
FIGURE 4.

Vδ usage by Vγ4+ cells in CIA animals. Lymph nodes were analyzed by flow cytometry as for Fig. 2⇑. Cells were triple stained for γδ TCR, Vγ4, and either Vδ4 (A), Vδ5 (B), or Vδ6.3 (data not shown) and the percentage of each Vδ subset was determined. The Vγ4/Vδ4+ and Vγ4/Vδ5+ subsets (circled populations) were then stained intracellularly for IL-17. Vγ4/Vδ6.3+ cells represented <0.5% of the Vγ4+ population and did not produce IL-17 (data not shown).

FIGURE 5.
FIGURE 5.

Vγ4 (A) and Vδ4 (B) sequences from CIA-elicited γδ T cells. Vγ4 (C) and Vδ4 (D) sequences from naive γδ T cells.

Vγ4+ γδ T cells are pathogenic

To determine the contribution of the Vγ1+ and Vγ4+ subsets to the development of CIA, mice were injected i.v. on day 17 with an anti-Vγ4 mAb or anti-Vγ1 mAb, to deplete the Vγ4+ or Vγ1+ subset, respectively, before the second injection of collagen/CFA. A control group of mice, injected with hamster IgG, was included in each experiment. Less than 5% of the Vγ1+ cells and 1% of the Vγ4+ cells remained detectable in the blood after depletion with the appropriate Ab (Fig. 6⇓A). As shown in Fig. 6⇓B, Vγ4-depleted mice had significantly less clinical disease as compared with control mice. In contrast, clinical disease scores were not significantly changed in Vγ1-depleted mice (Fig. 6⇓C). The overall incidence of disease was also lower in the Vγ4-depleted mice but not in the Vγ1-depleted animals (Fig. 6⇓D). On day 41, the mice were sacrificed and the joints from the Vγ4-depleted, Vγ1-depleted, and hamster IgG-treated mice were examined for changes in inflammation, pannus, cartilage damage, and bone damage. The Vγ4-depleted mice showed a statistically significant decrease (42%) in total score for all histological parameters examined when compared with those obtained from hamster IgG-treated mice (Table I⇓). In agreement with the overall disease scores, Vγ1-depleted mice showed no statistical difference in any of their histological scores when compared with control mice (Table I⇓).

FIGURE 6.
FIGURE 6.

A, The effect of Ab treatment on γδ T cell subsets. On day 17, mice were given either anti-Vγ4 Ab (n = 30) vs hamster IgG (hIgG) (n = 26) i.v. (B) or anti-Vγ1 Ab (n = 30) vs hIgG (n = 25) (C). The majority of the appropriate subset is depleted from the peripheral blood on day 21. A compensatory increase in the percentage of the other subset is seen. Mice were injected with collagen/CFA on days 0 and 21 and clinical disease activity in the mice was followed over time. Values represent the mean ± SEM of two separate experiments (maximum disease severity score = 16). **, p < 0.01; ***, p < 0.001. Statistical significance was determined using the Mann-Whitney U test. D, Incidence of disease for each of the groups as described in A and B. The incidence of disease for each hIgG-treated group was pooled. Statistical significance was determined using the log-rank test.

View this table:
Table I.

Histopathology scores in mice with CIA treated with either an anti-Vγ4 Ab or an anti-Vγ1 Ab

Next, total IgG, IgG1, and IgG2a anti-collagen Ab levels were measured in the sera from the treated and control mice to determine whether γδ T cell subsets contributed to anti-collagen Ab production. No anti-collagen Abs were detectable on day 0. On day 21, 4 days after the anti-Vγ4 or anti-Vγ1 treatment was given, the levels of total IgG, IgG1, and IgG2a anti-collagen Abs were still equivalent to those seen in hamster IgG-treated control animals. However, by day 41 there was a significant decrease in the total IgG and pathogenic IgG2a levels of anti-collagen Abs in the Vγ4-depleted mice (Fig. 7⇓A). In contrast, mice depleted of Vγ1+ cells showed no significant change in Ab levels (Fig. 7⇓B). The level of IgG1 anti-collagen Abs did not differ from control groups in either Vγ4-depleted or Vγ1-depleted animals.

FIGURE 7.
FIGURE 7.

Anti-collagen Ab levels in mice with CIA. A, Mice depleted with an anti-Vγ4 Ab (n = 30) were compared with hIgG-injected controls (n = 26). Mean ± SEM is shown for total IgG, IgG1, and IgG2a Abs. The data represent two separate experiments. *, p < 0.05; ***, p < 0.001. B, Same as in A except mice depleted with an anti-Vγ1 Ab (n = 30) were compared with those treated with hIgG (n = 25). As in A, mean ± SEM is shown for two separate experiments.

Vγ4/Vδ4 γδ T cells are not collagen specific

Most of the molecules identified so far as ligands for γδ TCRs, including T22b, appear to be host-encoded molecules whose expression is induced by inflammation or stress (reviewed in Ref. 36). The observed expansion of Vγ4/Vδ4+ cells in our model of CIA could be a response to CII, or be specifically induced by the arthritis, or by treatment with CFA, which is used in the immunizations. To examine this, mice were immunized with PBS/CFA, which does not cause CIA in DBA/1 mice. Similar to CII-injected mice, the total number of γδ, Vγ1+, and Vγ4+ T cells increased after each PBS/CFA injection (Fig. 8⇓, A and B) and moreover, the Vγ4+ subset showed the same activated phenotype (high CD44, low CD62L, and low CD45RB expression) as in collagen/CFA-treated mice (Fig. 8⇓C). However, the timing of the response was different. Despite similar initial responses, the maximal response measured by the percentage of activated Vγ4+ cells after the second PBS/CFA immunization was delayed, peaking at 6 days vs 4 days in CIA mice, perhaps reflecting less inflammation in PBS/CFA-treated mice than in mice with CIA. As in CIA mice, the responding Vγ4+ γδ T cells were preferentially paired with Vδ4 (Fig. 8⇓D). Because the response is independent of both CII and arthritis, the ligand that drives expansion of the Vγ4/Vδ4+ γδ T cell subset during CIA cannot be collagen nor depend upon a host ligand induced specifically during arthritis. Instead, the ligand is most likely driven by the host response to the inflammation caused by CFA or to CFA itself.

FIGURE 8.
FIGURE 8.

The total numbers of γδ T cells, Vγ1+ cells, and Vγ4+ cells obtained from mice that had received PBS/CFA injections on days 0 and 21 (black arrows). On various days, the draining lymph nodes (inguinal, brachial, and popliteal) were removed and cells were stained for γδ T cell subsets. Using FACS analysis, the total number of γδ cells (A) and individual subsets (B) was calculated. Each time point represents the average + SEM for at least four different mice. C, On designated days after collagen/CFA injections (black arrows), γδ T cells were stained for Vγ1 and Vγ4 expression and for levels of CD62L, CD44, or CD45RB. The mean percentage + SEM of cells having an activated phenotype (CD62Llow, CD44high, CD45RBlow) is shown. D, The Vδ4 usage by Vγ4+ cells in PBS/CFA-injected animals. Lymph nodes were analyzed by flow cytometry and cells were triple stained for γδ TCR, Vγ4, and Vδ4 to determine the percentage.

Discussion

Several of our findings imply that Vγ4+, but not Vγ1+, cells are pathogenic in CIA. First, although Vγ1+ and Vγ4+ cells both increased during CIA after the second collagen/CFA injection, the Vγ4+ cells increased more rapidly, and to a greater extent, than the Vγ1+ subset. As well, many of the Vγ4+ γδ T cells expressed markers of activation following the collagen/CFA injections. Second, the Vγ4+ subset predominately produced IL-17, which is associated with inflammatory damage in CIA. Finally, depletion of the Vγ4+ cells before the second collagen/CFA injection led to a decrease in the severity and incidence of CIA.

In contrast, the Vγ1+ cells appeared unresponsive during induction of CIA. However, because surface markers of activation have not been studied extensively on γδ T cells, it is possible that characteristics of activation for Vγ1+ cells are different. Therefore, the removal of Vγ1+ cells at a different time point of the disease process could potentially uncover a role for this subset as well.

Although the function of γδ T cells often segregates with their Vγ chain usage, a recent study demonstrated unusual TCR requirements for the recognition of a certain ligand. Here, Shin et al. (35) found that a γδ T cell clone, G8, recognized its ligand, T22b, almost exclusively through the Dδ2 portion of the δ-chain. Using a T22b tetramer to identify T22b-specific γδ T cells, they found that a particular motif in the δ-chain CDR3 was sufficient to confer T22b binding. This motif was generated by the use of Dδ2 in only one of three possible reading frames (encoding (S)EGYE), flanked by two other conserved residues, a preceding tryptophan encoded by either the 3′ end of Vδ6.3 or by Dδ1, and a following leucine encoded by N- or P-nucleotide additions. A variety of CDR3δ lengths were permitted among T22b-binding γδ TCRs, and the required motif could be generated almost entirely from germline-encoded components.

Our findings suggest that γδ T cells bearing particular TCRs are preferentially expanded by an Ag present during CIA. However, the requirements for binding this putative Ag appear to include elements of both the γ- and δ-chains, because activated cells expressing the Vγ4/Vδ4 combination predominated. We also found a highly predominant recurrent motif in the CDR3 regions of the TCR δ-chain, including a single reading frame for Dδ2 among all CIA-elicited Vδ4s ((I)GGIR) (30 of 30 clones). Although the (I)GGIR reading frame is normally somewhat more common than the (S)EGYE reading frame, only 5 of 13 clones derived from naive mice used the (I)GGIR reading frame (Fig. 5⇑D). The Dδ2 was preceded by an arginine in 27 of 30 clones, encoded by either Vδ4 or N/P nucleotides, which may explain the preference for this Vδ. Also, a second arginine, encoded by the 3′ end of Dδ2, was found in all 30 CIA-elicited Vδ4 clones, compared with only 4 of 13 naive clones. Unlike the T22b-reactive δ-chains, the lengths of the CIA-elicited δ-chain CDR3s also seemed to be restricted, ranging between 5 and 6 aa between V and J in 23 of 30 clones.

The CDR3 of the CIA-elicited Vγ4s was also very limited. A total of 37 of 42 clones contained only a single amino acid, leucine, between V and J, and four of the six possible leucine codons were found, consistent with the selective expansion of Vγ4/Vδ4+ cells bearing a particular motif in the γ-CDR3 as well. This contrasts markedly with the findings for T22b-binding γδ TCRs, in which the γ-chain appeared to be uninvolved in ligand interaction (35). Indeed, the γδ TCR restrictions associated with the CIA-selected γδ T cells are reminiscent of those common for αβ TCRs specific for a given ligand. Therefore, γδ T cells in the CIA model appear to be selected in a manner different from the T22b-binding cells, and more akin to the selection of αβ T cells. Thus, the question of whether γδ TCR ligand recognition differs fundamentally from that of αβ TCRs remains open.

The response of this Vγ4/Vδ4+ γδ T cell subset during CIA does not require immunization with CII, or the induction of arthritis, but instead can be elicited (with slightly different kinetics) by CFA emulsified in PBS only. This suggests that a host ligand induced by CFA treatment, or a ligand within CFA itself, stimulates the response of Vγ4/Vδ4+ γδ T cells. The independence of the Vγ4/Vδ4 response to CII and arthritis shows that these cells are not sufficient for the development of arthritis. However, many autoimmune models require adjuvant to initiate disease and our findings show an important contribution from this γδ T cell subset in the outcome of CIA. In fact, one might speculate that in individuals with RA, natural adjuvants (e.g., bacterial or viral infections) stimulate γδ T cells, which then change the cytokine environment and alter the immune balance, exacerbating disease.

Opposing roles for Vγ1+ and Vγ4+ cells in various disease models have been previously noted. For example, Vγ4+ cells suppress allergic airway hyperresponsiveness (37) whereas Vγ1+ cells enhance airway hyperresponsiveness (38). In addition, Vγ4+ cells promote myocarditis in a coxsackievirus B3 model, whereas Vγ1+ cells are protective (39). This difference was attributed to skewing of the Th1/Th2 αβ T cell response by the γδ T cells. Interestingly, when using the BALB/c mouse in an effort to look at IL-4-producing CD4+ cells, infection with a strain of coxsackievirus B3 that promotes myocarditis resulted in an expansion of Vγ4/Vδ4+ cells (40). Intracellular cytokine staining of these Vγ4/Vδ4+ cells revealed that a large proportion (50%) produced IFN-γ (IL-17 was not measured). In our model of CIA, only 4% of the Vγ4/Vδ4+ cells produced IFN-γ (data not shown). These results suggest Vγ4/Vδ4+ cells have the potential to produce Th1 and/or Th17 cytokines, and differences in the disease models may determine which type of cytokine is produced.

γδ T cells have been previously identified as a source of IL-17. Stark et al. found that in B6 mice, both αβ and γδ T cells produced IL-17 (41). Similarly, both Lockhart et al. (42), studying Mycobacterium tuberculosis infection of the mouse lung, and Umemura et al. (43), studying pulmonary M. bovis bacilli Calmette-Guérin infection, found that γδ T cells were in fact the dominant source of IL-17, rather than CD4+ αβ T cells. Finally, resident Vδ1+ γδ T cells have been shown to rapidly produce IL-17 in response to Escherichia coli infection and be critical for the neutrophil response to the infection (44). Our results showed that in CIA, Vγ4/Vδ4+ cells predominated and the vast majority of these cells in both the draining lymph node and the joints of mice could be stimulated to produce IL-17. Moreover, there were as many IL-17+ Vγ4+ cells in the lymph node as CD4+ αβ TCR+IL-17+ cells. Based on studies in RA and experimental allergic encephalomyelitis, IL-17 is now considered a major player in chronic autoimmune diseases. Studies in CIA have shown that disease is markedly suppressed in IL-17 “knocked-out” mice and IL-17 is responsible for the priming of collagen-specific T cells and for collagen-specific IgG2a Ab production (45). In addition, neutralization of IL-17 after the onset of CIA reduces joint inflammation, cartilage destruction and bone erosion (46). Depleting Vγ4+ cells in our model and thus, removing a large source of IL-17, may explain why these mice had less severe arthritis and a lower incidence of disease.

In this study, we demonstrate an Ag-driven oligoclonal response by the Vγ4/Vδ4+ γδ T cell subset. These cells are a potent source of IL-17 in the lymph nodes and joints of CIA mice and contribute to disease development. Therefore, it may be possible to reduce chronic inflammation in diseases such as CIA by preventing or eliminating the response of certain subsets of γδ T cells. The effect that γδ T cells have on αβ T cells, NK cells, B cells, and other immune cells in this disease model still needs to be defined. Better understanding of the contribution of γδ T cells to the pathogenesis of autoimmune and allergic diseases may lead to therapies that target this small population of cells.

Acknowledgments

We thank N. Banda (University of Colorado Health Sciences Center) for technical assistance with establishing the disease model and R. Kedl (National Jewish Medical and Research Center) for his constructive comments.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by an Investigator Award from the Arthritis Foundation (to C.L.R.), an award from the American Heart Association (to J.D.F.), and by National Institutes of Health Grants 2R01 A1044920 (to R.L.O.) and 2R01 HL65410 (to W.K.B.).

  • ↵2 Address correspondence and reprint requests to Dr. Christina L. Roark, Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, K409, Denver, CO 80206. E-mail address: roarkc{at}njc.org

  • ↵3 Abbreviations used in this paper: CIA, collagen-induced arthritis; RA, rheumatoid arthritis; CII, type II collagen.

  • Received May 4, 2007.
  • Accepted July 31, 2007.

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