Bone marrow macrophages process exogenous Toxoplasma gondii polypeptides for recognition by parasite-specific cytolytic T lymphocytes
Denkers, E.Y.; Gazzinelli, R.T.; Hieny, S.; Caspar, P.; Sher, A.
Journal of Immunology 150(2): 517-526
1993
ISSN/ISBN: 0022-1767 PMID: 8419484 Document Number: 408358
CD8+ T cells from mice vaccinated with an attenuated strain of Toxoplasma gondii have previously been shown to have cytolytic activity against bone marrow macrophages (BMM-vphi) preincubated with a soluble tachyzoite extract. In the present study, we show that class I-transfected L cells differ from BMM-vphi in that although both cell types are recognized CTL after infection, only BMM-vphi are killed after sensitization with soluble tachyzoite extract. Gel filtration studies indicated that the T. gondii Ag responsible for sensitization of BMM-vphi are macromolecules of M-r gtoreq 12,000. In contrast, peptides derived by tryptic digestion of this material were found to sensitize both transfected L cells and BMM-vphi. Although exogenous beta-2-microglobulin markedly enhanced peptide sensitization of BMM-vphi, no such effect was observed using the macromolecular preparation. This result suggests a requirement for cellular internalization in the processing by BMM-vphi of soluble Ag for class I-restricted recognition. In related experiments, infected and Ag-sensitized BMM-vphi were found to express cross-reactive T. gondii epitopes, as determined by cold target inhibition studies. Supernatant derived by 100,000 times g centrifugation of tachyzoite extract had potent sensitizing activity, and after anion exchange chromatography most of the activity was associated with a single fraction. The p30 Ag was not detected by immunoblot analysis in the biologically active supernatant and chromatographic fractions. These findings establish the feasibility of identifying the parasite Ag recognized by CD8+ effectors by direct fractionation of T. gondii proteins coupled with sensitization of BMM-vphi targets.