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Investigation of the effect of genetic polymorphism on the selection of antigenic peptides in major histocompatibility complexes

University of British Columbia

Major histocompatibility complexes (MHCs) play a prominent role in the human adaptive immune system by presenting peptides derived from both host and foreign sources on the cell surface to T cells and eliciting appropriate immune responses during pathogenic invasions. MHC genes are highly polymorphic and the effect of polymorphism on the phenotype, known as an individual’s immunopeptidome, is still unclear. In this thesis, two independent but complementary methods of research were conducted to better understand the interaction between MHC alleles and the identities of peptides presented. First, the antigen presentation machinery was reconstructed in vitro for class II MHCs. This was accomplished by cloning and expressing HLA-DM and HLA-DR in insect cells and purifying the proteins via affinity and size exclusion chromatography. While DM was successfully purified, DR was not. However, once established the in vitro system will offer a novel way to deduce the preferred binding residues for any MHC allele or combinations of alleles, information traditional immunoprecipitation experiments cannot obtain. Next, in an effort to achieve higher confident assignments of class II MHC binding residues, a cell surface acid elution protocol was developed and performed on consanguineous B cell lines. Extracted peptides were identified using liquid chromatography tandem mass spectrometry. To verify that most surface peptides originated from MHCs, lentiviral shRNA was used to knock down HLA-A prior to acid elution, and the identities of peptides were compared to those obtained from the same cell line transduced with a non-targeting shRNA sequence. Results followed anticipated trends and validated the technique as a means to extract MHC peptides. Furthermore, the nature of consanguineous data sets allows for intra-experimental comparisons to decipher allele-specific peptides. Ultimately, these experiments present new ways to study the immunopeptidome and possess the potential to be applied to the vaccine development research field in the future.

Text

2015-03-11

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/52349

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/