T cell development in thymus grafts in the absence of donor HSC engraftment. (A) Newborn wild-type thymi were transplanted into Rag2^−/−γ_c^−/−Kit^W/Wv recipients (n = 57), and analyzed 9–11 wk later. One thymus graft representative of sustained T cell development (left column), and the endogenous thymus of the same recipient (middle column) are shown. Rag1^−/− recipients (n = 3) were transplanted as controls and analyzed at 6 wk (right column). Dot plots display cells pregated for thymus donor (CD45.1⁺) or host origin (CD45.1^-), and stained for CD4 and CD8 expression. Numbers indicate percentages of cells in each quadrant. (B) CD45.1⁺ thymus grafts implanted into CD45.1⁻ Rag2^−/−γ_c^−/−Kit^W/Wv (n = 57) or Rag1^−/− (n = 7) recipients were analyzed 9–11 wk later by flow cytometry for the percentage of DP thymocytes (left), for the percentage of CD45.1⁺ donor cells per total cells (middle), and for absolute numbers (right) of CD45.1⁺ donor-derived thymocytes within each graft. Each symbol represents an individual lobe. (C–F) CD45.1⁺ wild-type mice (B6/Ly5.1), nontransplanted Rag2^−/−γ_c^−/−Kit^W/Wv mice, and a representative Rag2^−/−γ_c^−/−Kit^W/Wv recipient (n = 57) at 9–11 wk after thymus transplantation were analyzed for the presence of HSCs (Lin⁻Sca1⁺Kit⁺; C; gated on Lin⁻), T cells (CD3⁺CD19⁻), B cells (CD3⁻CD19⁺; D; total splenocytes), NK cells (CD3⁻NK1.1⁺), NKT cells (CD3⁺NK1.1⁺; E; gated on CD19⁻ splenocytes), and myeloid cells (CD11b⁺ cells expressing varying levels of Gr1; F; gated on CD4⁻CD8⁻CD3⁻CD19⁻Ter119⁻ splenocytes). Percentages shown in F next to the corresponding FACS plots refer to percentage of donor (CD45.1⁺) or host (CD45.2⁺) cells. Numbers indicate percentages of cells in each gate.