Introduction.

Cytotoxic T lymphocyte (CTL) responses have been shown to contribute to immunity against intracellular pathogens. However, no information is available on the role of CTL in immunity to bovine paratuberculosis, caused by the intracellular pathogen Mycobacterium paratuberculosis (Mp.). To study paratuberculosis specific CTL responses we aim to use the cross-priming abilities of heatshock protein (Hsp70) to transport antigen into MHC class I pathway of antigen presenting cells (APC). As a first step, the specific interaction of Mp. Hsp70 with APC is illustrated in the present study.

Material and methods.

Purified recombinant Mp. Hsp70 protein was conjugated to FITC to study binding and uptake of the Hsp70. FITC-conjugated BSA or OVA were used as a negative control. Unlabeled Hsp70, OVA and alpha2-macroglobulin were used in competition studies. Bovine monocytes were isolated by magnetic separation using CD14 labeled magnetic particles. The CD14 positive cells were cultured in culture medium, to obtain macrophages, or culture medium supplemented with bovine rIL-4 and rGM-CSF, to obtain dendritic cells. The murine RAW264.7 macrophage cell line was used for comparison. Cells were analysed using confocal microscopy, and flow cytometry.

Results.

When APC are incubated with Hsp70-FITC, a clear interaction with the protein can be visualized which can be specifically inhibited using alpha2-macroglobulin.

Conclusion.

Bovine APC clearly show interaction with Mp. rHsp70 molecules similar to that observed in murine model systems, indicating uptake also occurs via the alpha2-macroglobulin receptor. For the use of Hsp70 as a tool to induce and study bovine CTL responses, proof of receptor mediated uptake of rHsp70 by APC is a first important step.