Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers

We characterized antigen-specific CD4(+) T cells in six patients with treat ment-resistant Lyme arthritis, using an HLA-DRB1*0401 major histocompatibil ity complex (MHC) class II tetramer covalently loaded with OSPA164-175r an immunodominant epitope of Borrelia burgdorferi. Direct analysis of OspA-tet ramer binding CD4(+) cells in patients expressing the HLA-DRB1*0401 allele revealed frequencies of between <0.005 and 0.1% in peripheral blood (n = 6) , and between <0.005 and 3.1% in synovial fluid (n = 3). OspA-tetramer(+)CD 4(+) cells were directly cloned at 1 cell per well and expanded by mitogen and IL-2 on allogeneic feeder cells. As measured by [H-3]thymidine incorpor ation. 95% of 168 T cell clones from synovial fluid binding the OspA-tetram er were antigen-reactive. Clones generated from peripheral blood revealed a different pattern of responsiveness when compared with clones generated fr om synovial fluid, as measured by proliferation, IFN-gamma, and IL-13 secre tion. These clones, selected on the basis of their peptide binding, also re sponded to whole protein, but with a different cytokine profile. Our studie s demonstrate that MHC class II tetramers can be used in humans to directly identify, isolate, and characterize antigen-reactive T cells from an infla mmatory compartment.