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H2-M3wt-restricted, Listeria monocytogenes-specific CD8 T cells recognize a novel, hydrophobic, protease-resistant,
C Nataraj1, M L Brown, R M Poston
1Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Abstract:
Mice infected with Listeria monocytogenes (LM) generate H2-M3wt-restricted CD8 effectors which recognize a heat-killed LM-associated antigen (HAA) presented by macrophages. To characterize HAA, we extracted a bioactive component from LM using SDS or NaOH. Extracted HAA aggregated in hydrophilic solvents but dissociated in the presence of SDS into a smaller subunit which migrated in Sephadex G-200 between chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). HAA bioactivity and size was unaffected by proteinase K under conditions which degraded virtually all detectable protein. HAA was also unaffected by other proteases, RNase and DNase, but HAA bioactivity was destroyed by periodate, an agent that degrades carbohydrates. These studies demonstrate that H2-M3wt can present a hydrophobic, non-peptide, microbial antigen, probably glycolipid in origin, to CD8 T cells.
Insights
Mice immune cells recognize a heat-killed Listeria monocytogenes-associated antigen (HAA) presented by macrophages. This study reveals HAA is a non-peptide, likely glycolipid, antigen, not protein, recognized by CD8 T cells.
Area of Science:
- Immunology
- Microbiology
- Biochemistry
Background:
- Mice infected with Listeria monocytogenes (LM) develop CD8 T cell responses restricted by H2-M3wt.
- These CD8 T cells recognize a heat-killed LM-associated antigen (HAA) presented by macrophages.
Purpose of the Study:
- To characterize the biochemical nature of the H2-M3wt-restricted LM-associated antigen (HAA).
Main Methods:
- Extraction of HAA from LM using SDS or NaOH.
- Size exclusion chromatography (Sephadex G-200) to determine subunit size.
- Enzymatic degradation assays using proteinase K, other proteases, RNase, DNase, and periodate.
Main Results:
- HAA dissociated into smaller subunits in SDS, migrating between 12.5 and 25 kDa.
- HAA bioactivity and size were resistant to proteinase K and other proteases, RNase, and DNase.
- HAA bioactivity was abolished by periodate, indicating a carbohydrate component.
Conclusions:
- H2-M3wt presents a hydrophobic, non-peptide antigen to CD8 T cells.
- The antigen is likely glycolipid in origin.
- This finding expands the understanding of antigen presentation beyond peptides.