Bone marrow macrophages process exogenous Toxoplasma gondii polypeptides for recognition by parasite-specific cytolytic T lymphocytes.
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 phi) preincubated with a soluble tachyzoite extract. In the present study, we show that class I-transfected L cells differ from BMM phi in that although both cell types are recognized CTL after infection, only BMM phi are killed after sensitization with soluble tachyzoite extract. Gel filtration studies indicated that the T. gondii Ag responsible for sensitization of BMM phi are macromolecules of M(r) > or = 12,000. In contrast, peptides derived by tryptic digestion of this material were found to sensitize both transfected L cells and BMM phi. Although exogenous beta 2-microglobulin markedly enhanced peptide sensitization of BMM phi, no such effect was observed using the macromolecular preparation. This result suggests a requirement for cellular internalization in the processing by BMM phi of soluble Ag for class I-restricted recognition. In related experiments, infected and Ag-sensitized BMM phi were found to express cross-reactive T. gondii epitopes, as determined by cold target inhibition studies. Supernatant derived by 100,000 x 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 phi targets.
Denkers
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EY
Immunology and Cell Biology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Gazzinelli
R T
RT
Hieny
S
S
Caspar
P
P
Sher
A
A
D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology*; D001853:Bone Marrow / Q000502:physiology*; D005260:Female; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000276:immunology*; D015800:Protozoan Proteins / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014122:Toxoplasma / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000502:physiology
D000953:Antigens, Protozoan; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D015800:Protozoan Proteins; D001613:beta 2-Microglobulin