Effects of recipient pDCs on donor T-cell priming. (A) B6D2F1 mice were irradiated with 1300 cGy in 2 split doses of 650 cGy, 3 hours apart. At the time of the second irradiation, mice were injected with 1 mg of the pDC-depleting mAb 120G8 or isotype control antibody MAC49. Twenty-four hours later (the usual time of transplant), bone marrow and spleen were examined for pDC and cDC content. Anti–PDCA-1 (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD11c (Biolegend, San Diego, CA) mAbs were used for flow cytometric analysis. DCs were enriched using density-gradient centrifugation before examination.⁵  Representative plots shown. (B) B6D2F1 mice received TBI or 1 mg anti-NK1.1 as pretransplant conditioning, followed by 120G8 or control mAb. Purified carboxyfluorescein succinimidyl ester (CFSE)–labeled B6 CD45.1⁺CD4⁺ T cells were injected and proliferation of splenic T cells analyzed by CFSE dilution 3 days later. Cells were stained with CD45.1, CD4, and the vital dye 7-AAD. Histograms shown are gated on live CD45.1⁺CD4⁺ cells. No difference in alloresponse was observed in the absence of pDC in either the irradiated or nonirradiated setting. (C) Modfit software (Verity Software House, Topsham, ME) was used to quantify the extent of donor T-cell proliferation. Calculated proliferation index = Σ all cells/computed number of parent cells. Data shown representative of 3 experiments, with n = 12 and n = 7 in respective TBI and NK1.1 groups. P = .37 irradiated control mAb versus irradiated 120G8. P = .31 nonirradiated control mAb versus nonirradiated 120G8. *P < .001 irradiated versus nonirradiated control and 120G8 mAb treated.