Neutrophil-DC hybrids also function as APCs in living animals. (A) At 48 hours after thioglycollate treatment, C57BL/6 mice received intraperitoneal inoculation of OVA E. coli. One hour later, Ly6G⁺/CD11c^high/MHC II⁺ neutrophil-DC hybrids and Ly6G⁻/CD11c^high/MHC II⁺ traditional DCs were purified from the PECs and then cocultured with OT-II CD4 T cells (left panels) or OT-I CD8 T cells (right panels). The 2 populations purified from a second panel of mice receiving intraperitoneal inoculation of WT E. coli served as controls. Data shown are the levels (means ± SD from triplicate cultures) of ³H-thymidine uptake. (B) Ly6G⁺/CD11c^high/MHC II⁺ hybrids purified from GM-CSF–supplemented BM culture (day 6) were labeled with CFSE and then subcutaneously injected into C57BL/6 mice. Skin-draining LN samples harvested 48 hours later were examined for the presence of CFSE⁺ cells. Cryostat sections were examined under fluorescence microscopy after counterstaining with APC-conjugated anti-CD3 mAb (bar indicates 50 μm). (C-E) Ly6G⁺/CD11c^high/MHC II⁺ hybrids and Ly6G⁻/CD11c^high/MHC II⁺ traditional DCs purified from GM-CSF–supplemented BM culture from C57BL/6 (CD45.2) mice were pulsed for 30 minutes with OVA-coated latex beads (C) or BSA-coated beads (D). These APC preparations were then intraperitoneally injected into B6 SJL (CD45.1) mice that had received intravenous injection of CFSE-labeled OT-II CD4 T cells (CD45.2) 24 hours earlier. The recipient mice were analyzed 3 days later for the expansion of CD45.2⁺/TCR Vα2⁺ T cells in pancreatic LNs. Data shown are actual CFSE profiles (C-D) and the numbers (means ± SD from 3 mice per panel) of CFSE^lo T cells (E). (F-G) B6 SJL mice received systemic injection of anti–Gr-1 mAb or isotype-matched control IgG and intravenous injection of CFSE-labeled OT-II CD4 T cells both on day 0. (F) These mice (6 mice per panel) received intraperitoneal inoculation of OVA E. coli on day 1 and were examined for the expansion of CD45.2⁺/TCR Vα2⁺ T cells in pancreatic LNs. (G) Ly6G⁺/Ly6C⁺/CD11c^high neutrophil-DC hybrids or Ly6G⁻/Ly6C⁺/CD11c^high traditional DCs purified from the PECs from the second panel of thioglycollate-treated B6 SJL mice were intraperitoneally injected together with OVA E. coli to test their ability to restore the APC function in the previously discussed mice treated with anti–Gr-1 mAb. *P < .05; **P < .01 between the indicated samples. Data are representative of at least 2 independent experiments.