Targeting of anti–Siglec-H Ab to pDC precursors in vivo and priming of OVA-specific CTLs by sheep anti–Siglec-H–OVA immunoconjugate. (A) Mice were given intravenous injections of either sheep anti–Siglec-H IgG-FITC or, as a control, sheep anti–hSiglec-8 IgG-FITC. After 3 hours, bone marrow cells (top dot plots) and spleen cells (bottom dot plots) were isolated and labeled with anti-B220, anti-CD11c, and anti-Ly6C. The FITC-labeled cells were gated (boxes, left dot plots) and analyzed for expression of pDC markers B220, CD11c, and Ly6c. The percentage values are shown for each marker. (B) Anti–Siglec-H-OVA complex primes OVA-specific CD8⁺ T cells in vivo. C57BL/6 mice (n = 5) were primed by intravenous injection of anti–Siglec-H–OVA complexes or irrelevant complexes, in the presence or absence of CpG, and boosted after 7 days with vaccinia virus encoding full-length ovalbumin. CTL responses were assessed in the blood by FACS analysis using SIINFEKL-H2K^b tetramers 7 days after boosting. Mean proportions of tetramer-positive cells as a percentage of CD8 cells (± SEM) for each group are shown. The inset shows SDS-PAGE of sheep anti–Siglec-H IgG-OVA (imm-OVA) and preimmune IgG-OVA (preimm-OVA), together with the corresponding unconjugated IgGs (imm and preimm) run under nonreducing conditions. Molecular weight markers in kilodaltons are shown.