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The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex
A Jardim1, D L Tolson, S J Turco
1Department of Biochemistry and Microbiology, University of Victoria, B.C., Canada.
Insights
Leishmania donovani (LPG) protein components, not the glycan, stimulate T cell proliferation in immunized mice. This finding shifts understanding of immune responses to Leishmania donovani infection.
Area of Science:
- Immunology
- Parasitology
- Molecular Biology
Background:
- Leishmania donovani is a parasite that causes leishmaniasis.
- Lipophosphoglycan (LPG) is a major surface glycolipid of Leishmania parasites.
- The role of LPG in T cell stimulation has been previously investigated.
Purpose of the Study:
- To determine the specific component of Leishmania donovani lipophosphoglycan (LPG) responsible for T cell proliferation.
- To characterize the nature of the T cell response to LPG.
Main Methods:
- In vitro lymphocyte proliferation assays using purified LPG and its fractions.
- Flow cytometry to identify T cell subsets (CD4+, CD8+).
- Depletion of specific cell populations (Thy-1+ cells).
- Biochemical analysis including acid hydrolysis, trifluoromethanesulfonic acid hydrolysis, and reverse-phase chromatography.
- Mass spectrometry to determine molecular weight of protein components.
Main Results:
- Lymphocytes from LPG-immunized mice proliferated in response to LPG and its delipidated congener.
- T cell proliferation was dependent on dose and prior immunization.
- The proliferating T cell subset was identified as CD4+CD8-.
- T cell stimulation was associated with the core structure of LPG, and subsequent analysis revealed a protein component.
- The protein component, not the glycan, was responsible for lymphocyte stimulation.
Conclusions:
- The protein component of Leishmania donovani LPG is the primary stimulator of T cell proliferation.
- This finding challenges previous assumptions about LPG's role in immune responses.
- Understanding the protein component's role is crucial for developing effective vaccines or therapies against leishmaniasis.
Abstract:
Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan.