Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4 + CD25 + Foxp3 + T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen–specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that ( i ) B29 administration by itself suppressed disease, ( ii ) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and ( iii ) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

SmnmaryHeat-shock proteins have been shown to be critical antigens in a number of autoimmune diseases. In human arthritis and in experimentally induced arthritis in animals, disease development was seen to coincide with development of immune reactivity directed against not only bacterial hsp60, but also against its mammalian homologue. We have developed murine monodonal antibodies after immunization with recombinant human hsp60. Antibodies with unique specificity for mammalian hsp60, not crossreactive with the bacterial counterpart (LK1), and antibodies recognizing both human and bacterial hsp60 (LK2) were selected. Both antibodies recognize epitopes located between amino acid positions 383 and 447 of human hsp60. In immunogold dectron microscopy, the mitochondrial localization of hsp60 in HepG2 cells was shown. Furthermore, both LK1 and LK2 showed a raised level of staining in light microscopy immunohistochemistry of synovial membranes in patients with juvenile chronic arthritis. The increased staining for LK1, with a unique specificity for mammalian hsp60, thus unequivocally demonstrates that this is due to a raised level of expression of endogenously produced host hsp60 and not to deposition of bacterial antigens.

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Adjuvant arthritis (AA) can be induced in Lewis rats by immunization with mycobacterial antigens. Passive transfer of a T cell clone recognizing the 180-188 amino acid sequence in mycobacterial heat shock protein 60 (hsp60) was found to induce AA. In the present study, we investigated whether tolerance was obtained for this AAassociated T cell epitope after intranasal or s.c. administration of a peptide containing this epitope. Two 15-mer peptides containing the mycobacterial hsp60 sequences 176-190 and 211-225 were used; 176-190 contained the T cell epitope 180-188, which was recognized by the arthritogenic T cell clone A2b and was the immunodominant hsp60 T cell epitope after induction of AA, and 211-225 contained a T cell epitope that was recognized both after induction of arthritis with whole Mycobacterium tuberculosis and after immunization with mycobacterial hsp60. In rats treated intranasally or subcutaneously with 176-190 and immunized with mycobacterial hsp60, proliferative responses to 176-190 were reduced. Proliferative responses to 211-225 and to whole mycobacterial hsp60 were not affected. AA was inhibited intranasally in the 176-190-treated rats but not in the 211-225-treated rats.