Myeloid Leukemic Blasts Use the Endogenous HLA Class I Antigen Processing Machinery for Peptide Loading Onto HLA Class II Molecules: Implications for Immunotherapy.

Abstract 3114

Poster Board III-51

Besides professional antigen-presenting cells, certain tumor cells also have an intracellular machinery to process antigenic peptides for presentation via HLA class II molecules to CD4⁺ T cells. During the classical HLA class II presentation pathway, the Invariant Chain (Ii) stabilizes HLA class II molecules in the endoplasmic reticulum (ER) and mediates their transport to specialized lysosomal antigen-loading compartments termed MIICs. In HLA class II-transfected tumor cells however, it has been showed that HLA class II-peptide complexes can also be presented on the plasma membrane in the absence of Ii. The current study explores this alternative presentation pathway in physiologically HLA class II-expressing leukemic blasts and focuses on the role of endogenous antigen processing. We transduced the human KG-1 myeloid leukemic cell line with specific retroviral Ii siRNAs to evaluate the dependency of HLA-DR processing on the function of Ii. Western blot and flow cytometric analyses demonstrated that total and plasma membrane HLA-DR expression levels remained unaffected after Ii silencing in KG-1 blasts. Since HLA-DR expression does require peptide binding, Ii-independency in these blasts might be achieved by endogenous peptide loading in the ER. To test this hypothesis, we studied whether components of the endogenous HLA class I antigen processing machinery, including the proteasome and transporter associated with antigen processing (TAP), were involved in HLA-DR processing and presentation. Cytoplasmic degradation of endogenous antigens into peptides was suppressed by using the proteasome inhibitors bortezomib and MG-132. Additionally, retroviral transductions were performed with viral UL49.5 and BNLF2a proteins that interfere with the function of TAP in order to block the supply of proteasome-generated endogenous peptides into the ER. In both experimental setups, not only strong reductions in HLA class I, but also in HLA-DR expression levels were observed at the KG-1 plasma membrane, as determined by flow cytometry. Moreover, HLA-DR expression was completely abolished after dual suppression of TAP and Ii, which indicates that HLA-DR processing and presentation in leukemic blasts is dependent on either of these two molecules. Indeed, Ii silencing in blasts of the TAP-deficient human Kasumi-1 myeloid leukemic cell line resulted in a total loss of HLA-DR expression. Treatment of TAP-deficient Kasumi-1 blasts with proteasome inhibitors did not influence HLA-DR expression levels, confirming that the involvement of endogenous antigens in HLA-DR processing and presentation is related to the function of TAP. In conclusion, our data showing that KG-1 blasts are able to process antigens via the proteasome- and TAP-dependent pathway for HLA class II-restricted presentation and their known capacity to express endogenous peptides makes it likely that these tumor cells present tumor-associated peptides on both HLA class I and II molecules. Thus, the use of strategies that specifically target the HLA class I antigen processing machinery may represent an attractive immunomodulatory approach to simultaneously optimize HLA class I- and II-restricted presentation of relevant leukemia-associated peptides on either proteasome- or TAP-deficient leukemic blasts, thereby generating more effective whole-cell vaccines for the treatment of AML.