The Mechanisms of cross-presentation of a long-lived viral protein
Final Report Abstract
Cross-presentation is the pathway involved in processing of exogenously acquired antigens for presentation on MHC class I molecules on the cell surface. Cross-presentation can be performed by macrophages, dendritic cells (DC) or endothelia and is essential for the anti tumor response and for the immune response lo pathogens which do not infect dendritic cells. Previously, we have shown that for. cross-presentation of the nucleoprotein (NP) of lymphocytic choriomeningitis (LCMV) it is necessary that the very long lived, appropriately folded NP is expressed in antigen donor cells if DC, which take up the donor cells, shall successfully cross-present the NP. Fragments of NP or newly synthesized NP polypeptides do not sustain cross-presentation efficiently. NP cross-presentation in the in vitro system established in our laboratory requires the activities of lysosomal proteases and the proteasome. Moreover, inhibitors of heat shock protein 90 family proteins interfere with cross-presentation. In this project we have further investigated important parameters of crosspresentation. We have shown that the depletion of NP from lysates of antigen donor cells abrogates cross-presentation whereas the depletion of HSP90 or HSP70 do not affect crosspresentation. This suggests that HSP90 is required for cross-presentation in the antigen presenting cell but not in the antigen donor cell. We established the purification of HSP90 in our laboratory and showed that HSP90 from NP-deficient cells does not inhibit crosspresentation of lysates from NP expressing cells. Previously, it has been shown that the type I interferon IFN-a enhances cross-priming. We have shown here that the treatment of antigen donor cells with IFN-a and in particular the joint treatment with IFN-y and TNF-a enhanced NP cross-presentation in vitro. An interesting model for cross-presentation is the uptake of biodegradable PLGA mircospheres (MS) containing antigen and adjuvants as this vaccine formulation affords very efficient cross-priming leading to potent cytotoxic T lymphocyte (CTL) responses and eradication of large tumor masses in mouse lumor models. We investigated which type of antigen presenting cells account for MS cross-presentation in vivo and in vitro. We could show that both DC and macrophages can cross-present MS encapsulated ovalbumin (MS- OVA) but only DC also presented MS-OVA on MHC II molecules. Both CD8+ and CDS-DC from spleen were able to efficiently cross-present MS-OVA. Neither the in vivo depletion of CDI Ic+ DC nor depletion of macrophages interfered with MS-OVA cross-presentation while the simultaneous depletion of both populations abrogated cross-presentation of MS-OVA. Thus macrophages can sustain cross-priming of MS encapsulated antigen in vivo. In further subprojects on the role of proteasome activity for direct presentation of LCMV antigens on MHC I we could show that the treatment of mice with the clinically applied broad spectrum proteasome inhibitor bortezomib leads to a reduction of antigen presentation of class I restricted epitopes, to a reduced CTL response to LCMV, and to an increase in viral titers. We conclude that the treatment of multiple myeloma patients with bortezomib may enhance the susceptibility to viral infection. During further work with the LMP7 specific inhibitor PR-957 we could show that the immunoproteasome qualifies as a drug target for the treatment of autoimmune diseases like rheumatoid arthritis, inflammatory bowel disease, diabetes, and multiple sclerosis. PR-957 suppresses the production of proinflammatory cytokines like IL-23, IL-6 and TNF-a and interferes with Thl7 differentiation. These data, recently published in Nature Medicine and reviewed in Nature Reviews Immunology (see attached publications), identify the immunoproteasome as a very promising drug target for the treatment of autoimmune diseases.
Publications
- A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat. Med. 15, 781-787 (2009)
Muchamuel, T.
- The proteasome inhibitor bortezomib enhances the susceptibility to viral infection. J. Immmunol. 183, 6145-6150 (2009)
Basler, M., Lauer, C , Beck, U. & Groettrup, M.
