Antigen Cross-Presentation Allows the PR1 Leukemia-Associated Antigen To Be Processed from Both Proteinase 3 and Neutrophil Elastase to Prime T Cells.

Abstract

We have shown that the HLA-A2-restricted PR1 leukemia-associated peptide antigen is processed and presented endogenously to cytotoxic T lymphocytes (CTL) from both proteinase 3 (P3) and neutrophil elastase (NE). It has recently been shown that antigen cross-presentation of exogenous proteins through the endogenous antigen presentation pathway is required to elicit naïve CTL responses, however. Professional antigen presenting cells (APC), such as dendritic cells (DC), are especially able to cross-present antigens and prime CTL immunity. Since DC express both P3 and NE, however, they could not be used to study whether PR1 can be cross-presented. Therefore, we used our P3- and NE-transfected HMy.C1R B cells (which normally do not express either P3 or NE) to determine whether PR1 could be cross-presented and its mechanism. P3- and NE-transfected HMy.C1R B cells express P3 and NE in the cytoplasm, as determined by immunofluorescence microscopy. Concentrated supernatants derived from P3- and NE-transfected B cells also contain abundant amounts of both proteins by Western blotting. To define the pathway of P3 and NE trafficking, we measured PR1-CTL proliferation after co-incubation with P3- and NE-transfected B cells that were previously treated with the proteosome inhibitor lactacystin (LAC), or the trans-Golgi network (TGN) inhibitor brefeldin A (BFA). Co-immunoprecipitation experiments were performed to further map intracellular location of the proteins. Both P3- and NE-transfected B cells treated with LAC showed a dose-dependent decrease in PR1-CTL proliferation. PR1-CTL proliferation was also abrogated by BFA. Next, we immunoprecipitated ubiquitin-conjugated proteins derived from P3- and NE-transfected whole cell lysates and immunoblotted with anti-P3 and anti-NE, respectively. By Western blot, using a blocking peptide to ubiquitin and the non-transfected B cells as controls, we found that both P3 and NE were conjugated to ubiquitin, which facilitates processing through the proteosome. To determine how P3 and NE might be shunted into the endogenous pathway, we immunoprecipitated P3- and NE-transfected whole cell lysates with antibodies to adaptor protein 3 (AP3) and the endoplasmic reticulum chaperone GRP94. We found both P3 and NE bound to both of these intracellular proteins. In contrast, U937 leukemia cells, which also express P3 and NE assessed by intracellular flow cytometry, did not secrete either P3 or NE into culture supernatants, assessed by Western blotting. Together, these results suggest that both P3 and NE, which are the source proteins of the PR1 leukemia-associated antigen, can be cross-presented to PR1-specific CTL by hematopoietic-derived APC, such as B cells, through the endogenous pathway involving ubiquitin conjugation, proteosome cleavage and ER to TGN trafficking to the cell surface to prime T cells. Leukemia cells, while susceptible to lysis by PR1-CTL, are likely incapable of priming naïve CTL since they lack exogenous production of either P3 or NE. These findings have important implications in the design of future immunotherapy strategies against leukemia.