Our results imply that Vγ4+, but not Vγ1+ cells, are pathogenic in CIA. First, although Vγ1+ and Vγ4+ cells both increased during CIA after the second 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, while the Vγ1+ cells appeared unresponsive. Second, depletion of the Vγ4+ cells before the second collagen/CFA injection led to a decrease in the severity and incidence of CIA, which was not seen following depletion of Vγ1+ cells. Finally, only the Vγ4+ subset produced IL-17, which is associated with inflammatory damage in CIA. Since 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. Moreover, removing Vγ1+ cells before induction of CIA or at another time point of the disease process might uncover a role for this subset as well.
While the function of γδ T cells often segregates with their Vγ chain usage, recent studies have demonstrated an important role for the TCRδ chain in ligand recognition. Shin et al. 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
. 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 antigen present during CIA. However, the requirements for binding this putative antigen appear to include elements of both
the γ and δ chains, because activated cells expressing the Vγ4/Vδ4 combination predominated. We also found a 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] (30/30 clones). Although the (I)GGIR reading frame is normally somewhat more common than the (S)EGYE reading frame, only 5/13 clones derived from naïve mice used the (I)GGIR reading frame (Supplementary Fig. 3
). The Dδ2 was preceded by an arginine in 27/30 clones, encoded by either Vδ4 or N/P nucleotides, which may explain the preference for this V delta. Also, a second arginine, encoded by the 3′ end of Dδ2, was found in all 30 CIA-elicited Vδ4 clones, compared to only 4/13 naïve clones. Unlike the T22b
-reactive δ-chains, the lengths of the CIA-elicited δ-chain CDR3s also seemed to be restricted, ranging between 5-6 amino acids between V and J in 23/30 clones.
The CDR3 of the CIA-elicited Vγ4s was also very limited. 37/42 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 interaction21
. 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.
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 in23
). However, the observed expansion of Vγ4/Vδ4+
cells in our model of CIA could be due to a response to the CFA, which is used in the immunizations. Therefore, we have examined mice immunized with PBS/CFA, which does not cause CIA in DBA/1 mice. Similar to CII-injected mice, the total number of γδ, Vγ1+
T cells increased after each PBS/CFA injection and moreover, the Vγ4+
subset showed the same “activated” phenotype (high CD44, low CD62L, and low CD45RB expression) as before (data not shown). 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 versus 4 days in CIA mice (data not shown), perhaps indicating that different stimuli are triggered in CIA than by PBS/CFA treatment alone. Hybridomas are now being produced to further investigate whether Vγ4/Vδ4+
cells respond to collagen, or another host-derived molecule that is induced by the immunization.
Opposing roles for Vγ1+
cells in various disease models have been previously noted. For example, Vγ4+
cells suppress allergic airway hyperresponsiveness (AHR)24
cells enhance AHR25
. In addition, Vγ4+
cells promote myocarditis in a coxsackievirus B3 model, whereas Vγ1+
cells are protective26
. 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+
. Intracellular cytokine staining of these Vγ4/Vδ4+
cells revealed that a large proportion (50%) produced IFNγ (IL-17 was not measured). In our model of CIA, only 4% of the Vγ4/Vδ4+
cells produced IFNγ (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.
Two recent papers previously identified γδ T cells as a potent source of IL-1728,29
. Stark et al. found that in B6 mice, both αβ and γδ T cells produced IL-17. More recently, Lockhart et al., studying Mycobacterium tuberculosis infection of the mouse lung, found that γδ T cells were in fact the dominant source of IL-17, rather than CD4+
αβ T cells29
. 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+
cells in the lymph node as CD4+
cells. Based on studies in RA and EAE, 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” mice30
, and neutralization of IL-17 after the onset of CIA reduces joint inflammation, cartilage destruction and bone erosion31
. 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 antigen-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. 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.