Immunogenicity of MHC class I binding V_H peptides. Murine DCs were generated using a modification of the method of Lutz et al, in which bone marrow mononuclear cells are treated with GM-CSF and matured with LPS.44 Mature murine DCs (1 × 105) were pulsed with either V_H peptide (50 μg) or β-gal peptide (as a positive control) and β₂-microglobulin (5 μg) for 2 hours. The peptide-pulsed DCs were washed, irradiated, and mixed with splenocytes at a DC-to-splenocyte ratio of 1:20 in the presence of IL-2 for 2 weekly in vitro stimulations. The number of antigen-reactive T cells was then assessed using an IFN-γ ELISPOT assay after an overnight restimulation in the presence of peptide. Spot numbers were automatically determined with the use of a computer-assisted video image analyzer. The numbers of antigen-reactive cells (per 5 × 10⁴ cells) induced by MHC class I binding V_H peptide-pulsed DCs were compared to those of non–peptide-pulsed DCs alone (bracket indicates a significant increase; P < .05). The results shown for the MHC class I binding V_H peptides are representative of 3 independent experiments except for that of peptides K9I, K9T, N9Y, and Y9Y, which are representative of 4, 2, 2, and 2 independent experiments, respectively.