In vivo activation of APCs with TLR agonists but not transfer of ex vivo–matured CD11c⁺ DCs augments the effector function of adoptively transferred T cells. (A-B) Effects of TLR agonists on DLI-mediated LH-GVH reactivity in 4-week-old chimeras. (Ai) Experimental schema; (ii) groups of 4-week-old C3H.SW→B6 full chimeras (constructed using non-TCD BM; n = 8-10 mice/group) received nothing, CpG1826 ODNs, 2 × 10⁷ splenocytes from C3H.SW mice, 2 × 10⁷ splenocytes from C3H.SW mice plus 2 injections of 1.5 × 10⁶ ex vivo–generated host or donor-derived BM DCs administered intraperitoneally or intravenously on days 0 and +7, or DLI plus CpG1826 ODNs, as schematized. Changes in donor CD8⁺ chimerism were measured longitudinally in peripheral blood (mean ± SEM). *P < .001 for all groups versus mice that received DLI plus CpG1826 ODNs. The low levels of donor T-cell chimerism prior to adoptive transfer represent the lag time required to achieve equilibrium of donor chimerism in blood in contrast to spleen in the same model.²⁹  (Bi) Experimental schema; (ii) groups of 4-week-old C3H.SW→B6 full chimeras (constructed using non-TCD BM; n = 8 mice/group) were left untreated, treated with imiquimod, received DLI in the form of 2 × 10⁷ splenocytes from C3H.SW donor together with 2 × 10⁶ splenic CD11c⁺ DCs, or were pretreated with imiquimod or vehicle followed by DLI, as schematized. Changes in donor CD8⁺ chimerism determined by serial tail bleeding are presented as a mean percentage of donor chimerism ± SEM. This experiment has been repeated 3 times. *P < .001 for all groups versus mice that received DLI plus imiquimod. (C-H) Topically applied TLR7 agonist directly influences the in vivo activations status of APCs and cellularity in draining CLNs. Imiquimod was applied to 4-week-old C3H.SW→B6.SJL full chimeras once a day for 2 days. The following day, CLNs were retrieved from imiquimod-treated or untreated chimeras and analyzed by flow cytometry. (C) Total numbers of CD11c⁺ DCs in the CLNs and their origin analyzed with 2-color flow cytometry using anti-CD11c⁺ and anti-CD45.1 fluorescein-conjugated–specific antibodies. (D) Expression of CD40 and CD86 on the surface of host and donor CD11c⁺ DCs retrieved from the CLNs of untreated and imiquimod-treated chimeras was analyzed using anti-CD11c–, anti-CD45.1–, anti-CD40–, and anti-CD86–specific antibodies. Data are presented using the FlowJo histogram overlay scaling option “unit distribution” in which area under each curve corresponds to all gated CD11c⁺ cells. Data show the expression of CD40 and CD86 on donor and residual host derived CD11c⁺ DCs in imiquimod-treated (solid line) and untreated (filled histogram) chimeras, and isotype control (dotted line). (E-F) The total number of host and donor-derived CD11c⁺ DCs expressing CD40 and CD86. Values represent the mean ± SEM for combined CLNs per chimera (n = 3-4 mice/condition). (G-H) The origin and total cell number of IKDCs and B cells in CLNs of imiquimod-treated and untreated chimeras was determined by flow cytometry using anti-CD11c–, anti-CD45.1–, anti-CD49b–, and anti-B220–specific antibodies. Values represent the mean ± SEM (n = 3-4 mice/condition).