IT but not intravenous injection of WT BM progenitors into nonconditioned ZAP-70–deficient mice results in long-term thymopoiesis. (A) CD45.2⁺ ZAP-70^−/− mice were injected with lineage negative (lin⁻) BM progenitor cells (2 × 10⁵) isolated from CD45.1⁺ WT mice by either intravenous (IV) or IT routes. Animals were killed 20 to 25 weeks later, and the percentages of CD45.1⁺ cells in the lymph nodes were assessed. Dot plots showing representative CD45.1 and CD3 staining of WT and ZAP-70^−/− mice reconstituted by intravenous and IT administration of BM progenitors are shown (top). The percentages of naive (CD44^intCD62L^hi), central memory (CD44^hiCD62L^hi), and effector (CD44^hiCD62L^lo) T cells in these mice were monitored by assessing CD62L and CD44 expression in gated donor CD3⁺ lymphocytes. Representative dot plots are shown, and the percentages of each population are indicated (bottom). (B) Representative dot plots showing the percentages of CD45.1⁺ cells in the thymi of CD45.1 WT and CD45.2 ZAP-70^−/− mice reconstituted by IV or IT injection of WT CD45.1⁺ progenitors. The accompanying graph shows the absolute numbers of CD45.1⁺ donor thymocytes in ZAP-70^−/− mice reconstituted by intravenously (n = 8) and intrathymically (n = 10) administered progenitors. ***P < .001. (C) CD4/CD8 profiles of thymic donor cells were assessed after gating on CD45.1⁺ thymocytes. Representative dot plots from a control WT mouse and intravenously and intrathymically reconstituted ZAP-70^−/− mice are shown. The percentages of cells in each gate are indicated.