We discovered that during the early immune contraction phase of an acute infection with lymphocytic choriomeningitis virus, 2D TCR–pMHC affinity of TCR transgenic P14 T cells increased more for cells from the splenic red pulp (RP) than the white pulp (WP). This difference was governed by regulatory T cells and TGF-β. The increased 2D affinity of RP cells correlated with their increased ability to kill target cells and to recognize a CTL escape epitope. Memory precursors from WP preferentially developed into long-term memory cells as compared to cells from RP, despite expression of the same memory markers.