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Functional relevance and structural requirements of peptide transport in a murine carcinoma cell line

University of British Columbia

Major Histocompatibility Complex Class I molecules mediate the presentation of endogenously synthesised antigens to the cytotoxic cells of the immune system. The expression of Class I molecules on the surface of most somatic cells, therefore, provides a mechanism by which the immune system can identify and destroy infected or cancerous cells. However, down-regulation of surface Class I expression is frequently observed in virally infected and cancerous cells, a situation that allows these cells to evade detection by the immune system. The study of cells exhibiting deficiencies in Class I expression has yielded many insights into the cellular processes involved in Class I restricted antigen presentation. In this study the antigen processing deficiency of the murine small cell lung carcinoma cell line CMT.64 is characterised. CMT.64 cells express greatly reduced levels of Class I on their cell surface and are unable to present viral peptides to cytolytic T lymphocytes, unless treated with interferon-γ. Although these cells synthesise reduced amounts of both the heavy chain and beta-2-microglobulin components of the Class I complex, these deficiences do not account for the failure to present viral peptides. In addition, CMT.64 cells fail to express the genes encoding the TAP1 and TAP2 components of the TAP-dependent peptide transporter. The restoration of expression of these genes is sufficient to restore presentation of endogenously synthesised antigens. While being unable to present antigens to cytotoxic T cells, CMT.64 cells are efficiently lysed by natural killer cells. This recognition is unaffected by TAP gene expression, stabilisation of Class I by peptide pulsing or interferon-γ treatment. The TAP-dependent peptide transporter plays a critical role in the assembly of Class I complexes in the endoplasmic reticulum. Current models propose that this transporter is composed of both the TAP1 and TAP2 proteins. However, some results suggest that alternative forms of the TAP transporter may be functional. Studies with the TAP2 deficient RMA-S cell line revealed viral presentation in the absence of a TAP heterodimer. In this study, the role of the TAP1 protein in RMA-S presentation is addressed using an antisense approach. This strategy indicates that the single subunit contributes to Class I expression. To further investigate this activity, the TAP deficient CMT.64 cell line was utilized. When expressed individually, both TAP1 or TAP2 proteins are capable of increasing surface Class I expression and presentation of viral and allogeneic peptides by CMT.64 cells. The detailed characterisation of the CMT.64 cell line provides novel insights into Class I assembly and antigen presentation. This work represents the first demonstration that the lack of expression of TAP genes by carcinoma cells is sufficient to inhibit antigen presentation by these cells. This information may be relevant in the development of strategies aimed at reducing tumour growth. The demonstration that alternative forms of the TAP transporter are functional revises the model of peptide transport into the endoplasmic reticulum. This result also provides an explanation for the basal level of peptide transport observed in other antigen processing deficient cells.

Thesis/Dissertation

application/pdf

2009-04-02

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http://hdl.handle.net/2429/6747

Zoology

University of British Columbia

UBCV

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