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Tracking of V8.2-Positive Encephalitogenic T Cells by Complementarity-Determining Region 3 Spectratyping and Subsequent Southern Blot Hybridization in Lewis Rats after Neuroantigen Sensitization¹

Hiroshi Sakuma^*,, Kuniko Kohyama^*, Youngheun Jee^2,* and Yoh Matsumoto^3,*

^* Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan; and Department of Pediatrics and Developmental Biology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR - and -chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use V8.2 TCR and clonal expansion of the V8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of V8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that V8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of V8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.

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