Timing of DLI administration but not the level of residual donor chimerism influences the fate of adoptively transferred T cells in MHC-matched, mHAg-mismatched chimeras. (A-B) Splenocytes (2 × 10⁷) from naive or presensitized B10.D2-Thy1.1 mice were CFSE-labeled before transfer to 8-week-old B10.D2→BALBc-CD45.1 full and BALB/c-CD45.1 + B10.D2→ BALB/c-CD45.1 mixed allogeneic chimeras. The same dose of CFSE-labeled splenocytes from BALB/cThy1.1⁺ mice was transferred to 8-week-old syngeneic BALB/c→BALB/c chimeras. Adoptively transferred T cells were analyzed using anti-Thy1.1–, anti-CD4–, and anti-CD8–specific antibodies. (A) Representative CFSE profiles of DLI-derived CD8⁺ T cells (day +29 after DLI) and gating used to delineate in vivo unproliferated CFSE^hi from slow-proliferating CFSE^slow and fast-proliferating CFSE^fast DLI-derived T cells in allogeneic and syngeneic chimeras. (B) CFSE dilution of DLI-derived CD4⁺ T cells in the spleen of the 8-week-old mixed (▪) and full (♦) chimeras on days +5, +14, and +28 after DLI administration was contrasted with adoptively transferred DLI-derived T cells in syngeneic chimeras (▾). Data are presented as a mean percentage of CFSE^hi CD4⁺Thy1.1⁺ ± SEM and represent one of 2 independent experiments (n = 3-4 mice/group/time point). *P < .05 (▪ versus ▾) and *P < .05 (♦] versus ▾). (C) Rapid conversion to full donor T-cell chimerism in peripheral blood of full chimeras that received presensitized DLI. Changes in donor T-cell chimerism were determined by serial tail bleeding and staining for Ly9.1 and lineage-specific markers (mean percentage donor chimerism ± SEM; n = 5 mice/group). *P < .001 (CD4⁺; ▪ versus □) and #P < .01 (CD8⁺; ♦ versus ⋄). (D-E) Timing of DLI but not transfer of host CD11c⁺ DCs influences clonal expansion of IFN-γ–secreting DLI-derived T cells. B10.D2→BALB/c full chimeras received DLI from B10.D2-Thy1.1 mice, 4 (▪) or 8 (♦) weeks after alloBMT. Additional groups of 4- and 8-week-old full chimeras received with DLI 2 × 10⁶ host CD11c⁺ DCs (▵, ○). Absolute numbers of IFN-γ–secreting DLI-derived CD4⁺ Thy1.1⁺ (D) and CD8⁺Thy1.1⁺ (E) T cells were determined in spleens on days +5, +14, and +28 after DLI. Mean ± SEM is shown; n ≥ 3 mice/time point and group. *P < .05 (▪, ▵ versus ♦; for both CD4⁺ Thy1.1⁺ and CD8⁺Thy1.1⁺). (F) Transferred host derived CD11c⁺ DCs persist in MHC-matched, mHAg-mismatched chimeras. A total of 2 × 10⁶ splenic CD11c⁺ DCs from BALB/c-CD45.1 mice and 2 × 10⁷ splenocytes from the B10.D2-Thy1.1 mice were injected intravenously to 4-week-old B10.D2→BALB/c chimeras. On days +1, +7, and +28 after transfer, the presence of host-derived CD11c⁺ DCs in the BM, CLNs, mesenteric LNs (MLNs), and SPL was determined by flow cytometry using anti-CD11c– and anti-CD45.1–specific antibodies. The absolute number of host-derived CD11c⁺ DCs that had homed to BM, CLNs, MLNs, and SPL was calculated by multiplying the percentage of CD45.1⁺CD11c⁺ DCs by the total number of cells.