CML-IFN-DCs treated with LPS are capable of inducing expansion of CML-specific CD8⁺ T cells. (A-B) Autologous T cells were cocultured with DCs generated from monocytes from CML patients as previously indicated. After 6 days of culture, phenotypic analysis of the proliferating T lymphocytes was performed by means of flow cytometry using fluorochrome-labeled antibodies to CD8 and CD4 antigens. (A) The morphologic parameters and the staining for CD8⁺ T lymphocytes (the differentiated/activated lymphocyte population is gated as R1). (B) The CD8/CD4 ratio of lymphocytes as generated in the different culture conditions. (C) CD8⁺ and CD4⁺ T cells from CML patients were magnetically sorted and cocultured with autologous DCs (generated with either GM/IFN-α or with GM/IL-4 in the presence or absence of LPS as described). On days 7 and 14, the cells were harvested, cultured, and restimulated with DCs previously thawed and generated as described above. Recombinant human IL-2 (hIL-2) was added to the cultures on day 5 after the first stimulation and on day 3 after each restimulation. On day 21, the lymphocytes were stimulated with autologous CD34⁺ cells for an additional 24 hours, and then CD8⁺ T cells were magnetically sorted by positive immunoselection and tested in an ELISPOT assay for the production of IFN-γ. LPS-treated IFN-DCs did not induce any generation of IFN-γ production by the autologous T cells when CD34⁺ cells from HLA-matched healthy individuals were used for the final stimulation. Representative data obtained with cells from 1 patient out of 3 are shown.