Characterization of Vaccinia virus alternative antigen transport and processing pathways for their presentation to cytotoxic CD8+ T lymphocytes

Resumo

CD8+ cytotoxic T lymphocytes (CTL) screen the surface of the cells in the organism in seek of abnormalities, which allows their correct elimination. This is possible because the cells in the organism process proteins, either self-proteins or pathogen-derived. This degradation generates peptides that are loaded and presented on class I major histocompatibility complexes molecules (MHC-I). The presentation of antigens through the classical pathway involves the transporter associated to antigen processing (TAP). This transporter allows peptidic antigens access the endoplasmic reticulum, where they are loaded onto MHC-I complexes. However, different epitopes that are presented in a TAP-independent manner have been described. We aimed to characterize the protease or proteases involved in the TAP-independent antigen presentation. Using CD8+ T lymphocytes purified from spleens from VACV-infected C57BL/6 mice and dendritic cells derived from bone marrow (BMDC), we studied the importance of the proteasome in TAP-independent VACV antigen presentation. Measuring CD8+ T lymphocyte activation by intracellular cytokine staining (ICS), we estimated that, after treatment of C57BL/6 and TAP1-/- VACV-infected BMDC with the proteasome inhibitor lactacystin (LC), the inhibition of the antigen presentation is strong and similar in both cases. Therefore, just like in wild type BMDC, proteasomes play a key role in TAP-independent VACV antigen presentation in BMDC. With the aim of characterizing alternative proteases involved in the antigen processing we studied, for eight peptides, the activation of monospecific CTL after VACV-infected BMDC stimulation. These BMDC were also treated with inhibitors of different proteases. We only observed an effect on antigen presentation after the treatment with the pro-protein convertases inhibitor dec-RVKR-cmk, which inhibited the presentation of one epitope. Once we acknowledged the existence of alternative MHC-I antigen presentation mechanisms, we wanted to know whether these had any physiological relevance on a VACV infection. For that, we inoculated C57BL/6 as well as TAP1-/- mice with synthetic peptide-loaded BMDC. After fourteen days, we infected the mice with VACV and measured the viral load at different days post-infection and in different organs. In spleens we observed a protective effect by the three peptides included in the study in C57BL/6 as well as in TAP1-/- mice. These results suggest that different cell types may have different alternative antigen processing pathways activated, being more relevant in some organs than in others. Also, they confirm the existence of previously described VACV antigen processing TAP-independent route