The present study shows that virus-specific CD8+ T cells are enriched in synovial fluid from individuals with inflammatory arthritis. In particular, CD8+ T cells specific for two epitopes from EBV lytic cycle antigens (GLCTLVAML from BMLF1 and RAKFKQLL from BZLF1) may be present at very high frequency within the joints of EBV-seropositive patients. In donor RhA5, staining with tetrameric HLA-peptide complexes showed that T cells specific for these two epitopes accounted for 17.6% of all CD8+ T cells within synovial fluid (over 106 cells in a single joint aspirate). The findings described are not specific for RA and we obtained similar results in a patient with psoriatic arthritis (NR2) and in patients with osteoarthritis (NR3 and NR4). We also found very large numbers of T cells specific for an epitope (NLVPMVATV) from the CMV tegument protein pp65 in one patient with RA, and clearly detectable populations of T cells specific for this epitope in three other patients with RA. We did not find high frequencies of T cells specific for an epitope (FLRGRAYGL) from the EBV latent protein EBNA3A or of T cells specific for an epitope (GILGFVFTL) from the influenza A matrix protein within SFMCs in the patients studied. Thus, the synovial T-cell population is not simply a 'concentrated' pool of peripheral memory T cells. Many factors may contribute to the enrichment of T cells specific for certain viral antigens within the joint; these include preferential migration of subsets of T cells, local stimulation within the joint and protection from apoptotic cell death within the joint.
T-cell migration to sites of inflammation is a carefully controlled process [27,28,29] and T cells with an activated and memory phenotype may be preferentially recruited to a site of inflammation. The experiments described here show that the antigen-specific T cells within PBMCs are relatively more common in the CD62Llo, CD45RO+ and CD45RA- compartments than in the CD62Lhi, CD45RO- and CD45RA+ compartments. Thus, selection for activated/memory T cells is likely to account at least partly for the observed enrichment of virus-specific T cells within the joints. Current experiments are aimed at investigating the importance of expression of integrins and chemokine receptors by T cells in the recruitment of virus-specific T cells into joints [27,30,31].
It is also possible that T cells are stimulated to proliferate within the joints. Preliminary experiments (unpublished data) suggest that a small proportion of CD8+ T cells (usually <5%), including some EBV-specific T cells, are in cell cycle within synovial fluid. the stimulus to proliferation might be the relevant viral antigen itself. CMV dna has been detected in rheumatoid synovium . Bcells as well as T cells are recruited into inflamed joints and, in EBV-seropositive individuals, a subpopulation of these Bcells will be latently infected with EBV. One might therefore expect to find this virus within inflamed joints. Although some early studies  found no evidence of EBV infection within joints, other reports [34,35] described detection of EBV DNA within the joints of patients with RA and one study  described the use of in situ hybridization to detect EBV-encoded small RNA1 and LMP1 transcripts in synovial lining cells from RA patients. Furthermore Koide et al  derived a fibroblastoid cell line that expressed EBV proteins from the synovium of a patient with RA. Very recently, Edinger et al  reported detection of EBV DNA within synovia from 10 out of 11 patients with RA. That study also provides evidence of transcription of EBV EBER1 and BZLF1 in samples of synovia from patients with RA and osteoarthritis. Thus, expression of BZLF1 within the joint may be stimulating T cells specific for the HLA-B8 restricted RAKFKQLL epitope from BZLF1. Transcription of BMLF1 has, however, not been detected within synovial tissue, suggesting that alternative mechanisms may be responsible for driving the proliferation of CD8+ T cells specific for the HLA-A2 restricted epitope (GLCTLVAML) from BMLF1. One possible alternative is that antigen-presenting cells such as dendritic cells may take up EBV antigens and subsequently be recruited to joints where they present epitopes from the EBV antigens by 'cross-presentation' . A second possibility is that the virus-specific T cells are being stimulated by cross-reactive self-antigens expressed within the joint. In favour of this is the fact that a subpopulation of T cells specific for the HLA-B8 restricted RAKFKQLL peptide epitope is able to cross-react with a self peptide from a serine-threonine kinase .
Relative resistance to apoptotic cell death is a further theoretical factor that could influence the frequency of the virus-specific T cells within the joint. Although there is evidence that T cells within the joint are protected from apoptosis by type 1 IFN  there is no obvious reason to believe that the T cells specific for the RAKFKQLL and GLCTLVAML epitopes should survive more efficiently than other CD8+ T cells.
The results of the ELISpot assays for IFN-γ release after incubation of SFMCs with peptide epitopes from EBV and of the assays for TNF-α release after incubation of SFMCs with COS cells transfected with HLA-A2 and CMV pp65 suggest that the T cells within the synovial fluid retain their capacity to secrete proinflammatory cytokines. Within the joint, the secretion of such cytokines could lead to activation of synoviocytes and hence to the maintenance of inflammation . Cell-cell contact between the activated virus-specific T cells within the joint and the synoviocyte population is a second mechanism whereby the virus-specific T cells might interact with the indigenous cells within the joint and contribute to the pathogenesis of inflammatory joint disease .
Importantly, these experiments show that large numbers of T cells within the joint are specific for epitopes from certain viral proteins. Many previous studies analyzed T-cell receptor use of T cells within the joint and found evidence of clonality, and concluded that the T cells are being driven by a specific self-antigen. Paliard et al  found that clonality was particularly marked within Vβ 14+ T cells within synovial fluid and that Vβ 14+ T cells were not well represented within peripheral blood of patients with RA. Those authors suggested that a superantigen might have caused activation of Vβ 14+ T cells, with recruitment of selected Vβ 14+ T cell clones into the joint followed by deletion of Vβ 14+ T cells in the periphery.
It will be interesting to analyze Vβ usage of our HLA-viral peptide tetramer-reactive CD8+ T cells. From the work we have described, it seems likely that at least some of the clonally expanded populations of CD8+ T cells found in synovial fluid are specific for viral antigens. The reasons for the presence of large numbers of synovial T cells specific for certain viral epitopes and not others remain unclear, and the role that these virus-specific 'bystander' T cells may play in the maintenance of inflammation needs further investigation.