Synergistic triggering of TLR7 and TLR9 or depletion of host T cells together with TLR9 activation is required to augment the DLI-mediated GVH reactivity in late full or early mixed MHC-matched, mHAgs-mismatched chimeras, respectively. (A-C) Effects of TLR agonists on DLI-mediated LH-GVH reactivity in 8-week-old full chimeras (constructed using non-TCD BM). (Ai) Experimental schema; (ii) 8-week-old C3H.SW→B6 full chimeras received either nothing or 2 doses of anti-CD25 mAb on days −7 and −4 prior to receiving DLI. Changes in donor T-cell chimerism were measured longitudinally in peripheral blood based on the differential expression of Ly9.1 on donor C3H.SW (Ly9.1⁺) and host (Ly9.1⁻) T cells. Data representing the mean percentage of donor chimerism ± SEM from one experiment are plotted as a function of time after DLI (at least 8-10 mice/group/time point). P =NS. (Bi) Experimental schema; (ii) 8-week-old C3H.SW→B6 full chimeras received either DLI in the form of 2 × 10⁷ C3H.SW splenocytes alone or were pretreated with imiquimod prior to DLI followed by CpG1826 ODNs as per schema. T-cell chimerism was determined by serial tail bleeding and staining for Ly9.1 and lineage-specific markers. Data are presented as the mean percentage of donor chimerism ± SEM (n = 8 mice/group). #P < .005 for CD4⁺ and *P < .05 for CD8⁺ T cells in full + DLI versus full + DLI + CpG + imiquimod. (C) Body weight changes of full chimeras that received either DLI alone (▪) or were treated with αCD-25 mAb (▿), imiquimod + anti-CD25 mAb (⋄) or imiquimod + CpG1826 (•). *P < .05 indicates weight change of more than 10% for (• versus all groups only at designated time points). (Di) Experimental schema; (ii-iii) B10.D2-Thy1.1 + BALB/c-CD45.1 (Thy1.2)→BALB/c-CD45.1 mixed chimeras received nothing (▾), DLI in the form of 2 × 10⁷ B10.D2-Thy1.1 splenocytes (⋄), anti-CD25 depleting mAb (□) or anti-Thy1.2 depleting mAb (•) on days −7 and −4 prior to administration of 2 × 10⁷ B10.D2 splenocytes, anti-Thy1.2 mAb, 2 × 10⁷ B10.D2 splenocytes, and CpG1826 ODNs on days 0, +3 and +7 after DLI (▿). Changes in donor CD4⁺ T-cell (ii) and granulocyte (iii) chimerism were determined by serial tail bleeding as described in Figure 1. P < .05 for mice that received DLI, anti-Thy 1.2 mAb, and CpG1826 CDNs versus other groups. Data represent the average percent chimerism ± SEM plotted as function of time after DLI (at least 5 mice/group/time point).