WT1 specific T cells persist in the periphery. (A) FACS analysis of peripheral T cells in spleen, LNs, and BM of A2Kb Tg mice killed 11 weeks after transplantation with TCR-transduced Lin⁻ A2Kb BM stem cells. Mice received untransduced stem cells (top row), WT1-TCR-transduced stem cells (middle row, n = 8), or LMP2-TCR–transduced stem cells (bottom row, n = 5). Viable lymphocytes were stained with anti-CD3, and anti–human Vβ2.1 (WT1-TCR) and anti–human Vβ13 (LMP2-TCR) antibodies before FACS analysis. Percentages of Vβ2.1⁺ and Vβ13⁺ cells in total CD3⁺ cells are indicated. (B) FACS analysis of splenocytes isolated from A2Kb Tg mice transplanted with Lin⁻ A2Kb BM stem cells transduced with the lentiviral WT1-TCR or LMP2-TCR vector. Splenocytes were stained with anti–murine CD3, CD4, and CD8 antibodies together with anti–human Vβ2.1 and anti–human Vβ13. (C) Cell surface CD3/TCR complex expression levels in peripheral T cells were determined by FACS analysis after staining with anti-CD3, and anti–human Vβ2.1 and anti–human Vβ13 antibodies. CD3/TCR expression levels of the WT1-TCR (self-reactive) and the LMP2-TCR (non–self-reactive) were compared with endogenous polyclonal T cells derived from transplanted stem cells (Vβ2.1⁻ and Vβ13⁻, respectively). Data are mean ± SD of CD3 mean fluorescence intensity (MFI; n = 8 mice for WT1-TCR and n = 5 mice for LMP2-TCR). *P < 0.05, 1-way ANOVA. **P < .01, 1-way ANOVA. (D) Modulation of TCR and/or CD8 expression was determined by FACS analysis of peripheral T cells of spleen, LNs, and BM stained with anti-CD8, and anti–human Vβ2.1 (WT1-TCR, n = 8) and anti–human Vβ13 (LMP2-TCR, n = 5) antibodies. After gating on viable CD3⁺ cells, percentages of TCR^hi and TCR^lo populations are indicated. All recipient mice were A2Kb and received TCR-Td A2Kb stem cells.