Significance Before invading the central nervous system, encephalitogenic T cells cross a series of microenvironments where they interact with local cells. T-cell activation was visualized by specific calcium signals using a combination of a genetic calcium reporter, Twitch1, and in vivo two-photon microscopy. In peripheral immune organs, short-lived calcium signaling indicated antigen-independent interactions. By contrast, in the CNS, saturated long-lived calcium signaling was induced by endogenous autoantigens presented by a subset of local antigen-presenting cells. Because T-cell trafficking is controlled at serial checkpoints, our findings may help to identify therapeutic targets for preventing CNS inflammation.