T cells in adjuvant arthritis

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4+ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134+ T cells showed a specific proliferative response to the diseaseassociated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4+CD134+ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134+ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4+ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development. Introduction In several autoimmune diseases, for example rheumatoid arthritis, the involvement of CD4+ T cells in disease induction has been suggested [1]. As a treatment strategy, the manipulation of CD4+ T cells by CD4-directed antibodies has therefore been studied extensively [2]. However, because anti-CD4 therapy targets the whole CD4+ population, CD4+ T cells not related to the disease or involved in disease regulation will also be affected. Ideally, only the auto-aggressive CD4+ T cells that are involved in the disease process should be targeted. Because for many human autoimmune diseases the exact antigens recognized by these cells are not known, a therapy would be favorable that specifically targets the auto-aggressive CD4+ T cells and does not depend on the definition of the crucial auto-antigen. Because auto-reactive CD4+ T cells become activated upon recognition of their cognate antigen in the periphery, they will be transiently marked by the expression of T cell activation markers. In this respect, CD134 (OX40 antigen) is an interesting candidate target molecule, because CD134 is expressed in vivo exclusively on activated CD4+ T cells (reviewed in [3]). In experimental autoimmune encephalomyelitis, a disease model for multiple sclerosis, it has been shown that CD134 is preferentially expressed on pathogenic CD4+ T cells that home to the target organ (namely the central nervous system) [4], and transiently marks the auto-aggressive T cells specific R604 AA = adjuvant arthritis; APC = antigen-presenting cells; Con A = concanavalin A; DiD = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4chlorobenzenesulfonate salt; FITC = fluorescein isothiocyanate; FUdR(-dP) = 5'-fluoro-2'-deoxyuridine (dipalmitate); HSP = heat shock protein; ILN = inguinal lymph nodes; mAb = monoclonal antibody; Mt = Mycobacterium tuberculosis; PBS = phosphate-buffered saline; PE = phycoerythrin; PerCP = peridinin chlorophyll protein; PLN = popliteal lymph nodes; s.c. = subcutaneously; SI = stimulation index; TCR = T-cell antigen receptor. Arthritis Research & Therapy Vol 7 No 3 Boot et al. R605 for myelin basic protein [5]. Moreover, in this T cell transfer model, depletion of CD134+ T cells with an anti-CD134 immunotoxin results in the amelioration of paralytic symptoms [6]. Interestingly, in patients with rheumatoid arthritis a high percentage of CD4+ T cells in synovial fluid express CD134 in comparison with peripheral blood T cells [6,7], suggesting that auto-aggressive CD4+ T cells may be transiently marked by surface expression of CD134 in arthritis too. Here, we investigated whether CD134 can be used as a target for specific drug delivery to activated auto-aggressive CD4+ T cells in arthritis. For this purpose, the rat adjuvant arthritis (AA) model was studied. In this model, a syndrome resembling rheumatoid arthritis is actively induced in Lewis rats after immunization with Mycobacterium tuberculosis (Mt) in adjuvant [8]. We first analyzed the CD134 expression on CD4+ T cells during AA, and investigated the presence of auto-aggressive T cells within the CD134+CD4+ T cell subset. We also studied drug delivery to CD134+ T cells both in vitro and in vivo using liposomes coated with a CD134-directed monoclonal antibody (mAb) as a drug targeting system. To investigate the possibility for therapeutic intervention in arthritis, antiCD134 liposomes were loaded with a cytostatic drug and administered early in actively induced arthritis. We show that CD134 can be used as a marker for activated auto-aggressive T cells early in AA, that targeting of these cells in vivo can be achieved with anti-CD134 liposomes, and that the course of AA could be affected with drug-containing anti-CD134 liposomes. Materials and methods Animals Male inbred Lewis rats were obtained from the University of Limburg (Maastricht, The Netherlands) and were used between 7 and 10 weeks of age. The animals were kept under conventional conditions and had access to standard pelleted rat chow and acidified water ad libitum. The Utrecht University Animal Ethics Committee approved all animal experiments.