i.v. infusion of native autoantigen or its altered peptide variants is an important therapeutic option for the treatment of autoimmune diseases, because it selectively targets the disease-inducing T cells. To learn more about the mechanisms and kinetics of this approach, we visualized the crucial initial effects of i.v. infusion of peptides or intact protein on GFP-tagged autoaggressive CD4 ⁺ effector T cells using live-video and two-photon in situ imaging of spleens in living animals. We found that the time interval between i.v. injection of intact protein to first changes in T cell behavior was extremely short; within 10 min after protein application, the motility of the T cells changed drastically. They slowed down and became tethered to local sessile stromal cells. A part of the cells aggregated to form clusters. Within the following 20 min, IFN-γ mRNA was massively (>100-fold) up-regulated; surface IL-2 receptor and OX-40 (CD 134) increased 1.5 h later. These processes depleted autoimmune T cells in the blood circulation, trapping the cells in the peripheral lymphoid organs and thus preventing them from invading the CNS. This specific blockage almost completely abrogated CNS inflammation and clinical disease. These findings highlight the speed and efficiency of antigen recognition in vivo and add to our understanding of T cell-mediated autoimmunity.