FIG. 4.

Formation of peptide/class I MHC complexes through cross-presentation requires Ig. (A to D) OVA-E. coli bacteria were opsonized with serum from wild-type (A), RAG1^−/− (B), or C3^−/− (C) mice or with RAG1^−/− serum depleted of complement by heat inactivation (HI) (D). Opsonized nonfluorescent E. coli bacteria were then added to MHC class II-EGFP-expressing DCs, and at 2 hours, DCs were fixed and stained for SIINFEKL/K^b complexes using 25D1.16 antibody. Only wild-type and C3^−/− serum-opsonized E. coli bacteria were processed sufficiently at this time to detect intracellular SIINFEKL/K^b staining. (E) Surface display of SIINFEKL/K^b complexes was analyzed by flow cytometry after coculture of OVA-E. coli with DCs for 2, 8, and 24 h. E. coli bacteria were opsonized as described for panel A and added to DCs. Opsonization using wild-type and C3^−/− sera yields surface display of SIINFEKL/K^b complexes in greater than 50% of DCs starting at 2 hours postuptake. These data show that while RAG1^−/− serum promotes phagocytosis, opsonization using RAG1^−/− serum does not yield significant SIINFEKL/K^b presentation (20% of DCs express SIINFEKL/Kb complexes at 2 hours postuptake, which is similar to what is found using heat-inactivated RAG^−/− serum).