Recognition of mycobacterial HSP65 in association with HLA-DR4 is not sufficient for autoreactivity

The mycobacterial heat shock protein 65, class II major histocompatibility complex molecule HLA-DR4, and T cells have been implicated in autoimmunity. However, there has been no direct demonstration of the recognition of the heat shock protein 65 by T cells in association with HLA-DR4. In this study, we established T cell lines and a large number of T cell clones from healthy subjects vaccinated with killed mycobacteria. Among these subjects, three were HLA-DR4 positive, and the T cell lines and clones from these subjects responded to the mycobacterial heat shock protein 65. HLA-restriction studies were done with well-characterized anti-HLA class I, anti-HLA-DR, and anti-HLA-DQ antibodies. Only anti-HLA-DR antibodies inhibited the antigen-induced response of the T cell lines and clones to heat shock protein 65. When HLA-DR-typed allogeneic cells were used as antigen-presenting cells, the T cell clones from only one of the individuals were found to be HLA-DR4 restricted. To identify the epitopes recognized by these T cell clones, synthetic peptides were synthesized covering the entire sequence of mycobacterial heat shock protein 65. When tested with the heat shock protein 65-reactive T cell clones, peptides from five different regions of heat shock protein 65 stimulated the T cell clones in association with HLA-DR4. However, it is difficult to predict the role of HLA-DR4-restricted mycobacterial heat shock protein 65 peptides in autoimmunity from our studies, because the T cell clones were established from a healthy donor, and the peptides belonged to the regions that do not share sequence homology between the mycobacterial and human heat shock protein 65 or other proteins.