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Immuno Biology Tutorial Week 3

To determine whether GILT (gamma interferon-inducible lysosomal thiol reductase) defined in humans is involved in reduction events. These reduction events occurwhen proteins internalized by APCs are exposed to the acidic/proteolytic environment of the endocytic pathway. This generates peptides that attach to MHC class II alpha Beta diners and the intrachaindisulfide bonds are reduced to help move along this process.
To do this the researchers used a knockout mouse and were able to find partial cDNAs encoding mouse GILT by using a dbEST database. Aftercloning the cDNA, they found that mouse GILT had about 70% amino acid sequence identity to human GILT.
They then cloned the mouse GILT cDNA and transfected embryonic stem cells so that they couldinject two ES clones in the host mother to create mosaic animals. They found that the homozygous knockout animals did not have GILT but the wild-type animals expressed it. Wild-type and GILT knockoutanimals showed no difference in numbers of CD4 positive cells which suggested GILT does not play a major role in positive selection.
To determine whether GILT was essential in antigen processing, theresearchers created GILT negative mice and exposed them to the antigen, Hen egg lysozyme (HEL). Gilt-negative mice responded one-tenth as much as the wild-type mice. To further study the response,splenic APCs from both GILT-negative and GILT-positive animals were used in in vitro assays. Using four different HEL-specific hybridomas, they found that three epitopes contained cysteine residues. Theresults suggested that two of the epitopes did not require reduction and unfolding before proteolytic processing but the others did. To determine whether prior reduction and unfolding of HEL couldmake up for the absence of GILT, they reduced and carboxymethylated HEL to block its cysteine residues. The results proved the prior theory of the role of GILT in mediating reduction.
Finally...