HLA class II presentation pathway is in part mediated by autophagy and requires lysosomal processing. For the induction of antigen-specific CD4⁺ T-cell responses by autologous DCs transfected with MUC1 RNA, human CD4⁺ T lymphocytes were isolated from PBMNCs using magnetic bead technology and coincubated with MUC1 RNA-electroporated DCs. The antigen specificity of the elicited CD4⁺-mediated immune response was assessed after 2 restimulations on day 20 after T-cell induction in a [3H]thymidine proliferation assay. Blocking of HLA class I and II was performed by incubating DCs with specific monoclonal antibodies against MHC-I and -II, respectively, to confirm the HLA class II restriction. To analyze the pathways involved in the presentation of MUC1-derived HLA class II epitopes, DCs were incubated with agents known to inhibit the proteasome (LLM, LLnL, lactacystin), the egress from ER (brefeldin A), lysosomal acidification (chloroquine), autophagy (3-MA, wortmannin), and endosomal proteases (leupeptin, cathepsin B inhibitor II). In addition, DCs were electroporated with ICP47 IVT to determine the possible role of a functional TAP. DCs electroporated with irrelevant Her-2/neu RNA were included as control. The statistical significance of the proliferation reduction compared with DCs incubated with dimethyl sulfoxide (DMSO) plus anti-MHC class I antibody was analyzed in a Student t test. **P < .001, *P < .002. Error bars indicate standard deviation.