Defining novel targets for immunotheraphy of human cancer

Doctoral thesis English OPEN
Nunes, Claudia

Cancer cells frequently exhibit defects in apoptosis, contributing to increased survival and resistance to chemotherapy. This can result from decreased expression of pro-apoptotic proteins such as Bax, through abnormal proteasomal degradation. The central hypothesis of this project is that this abnormal proteasomal degradation in cancer cells will result in the generation of peptides that bind to MHC class I molecules, and displayed at the cell surface for CD8+ T cell recognition. If this is correct, then T cells directed against Bax peptides should be able to recognize and kill human cancer cells. To test this hypothesis, candidate HLA-A2 binding peptide epitopes were identified from Bax and T cell immunogenicity tested using IFNy ELISpot assays. Positive T cell responses were detected against Bax peptides in 10/16 healthy donors. The specificity of a CD8+ T cell clone (KSIVB17) derived from one donor, was mapped to two similar peptide epitopes, Baxi36-144 (IMGWTLDFL) and Bax135144 (TIMGWTLDFL). In addition to recognition of peptide pulsed target cells, this clone was also able to recognise primary tumour cells from CLL (chronic lymphocytic leukaemia) patients, and kill cervical carcinoma and osteosarcoma cell lines. T cell reactivity correlated with abnormal proteasomal degradation of Bax in cancer cells. Attempts to generate Bax-specific T cells in CLL patients demonstrated that naive and memory T cell responses were compromised compared to healthy donors. This compromised T cell immunity appeared to result from increased expression of immunosuppressive molecules, increased frequency of Treg cells and a skewed memory T cell phenotype in CLL patients. Overall, this research supports the novel concept that proteins such as Bax, which are abnormally degraded in cancer cells, can be targets for CD8+ T cells. However use of Bax in immunotherapy will require strategies to overcome suppression of T cell responses in CLL and other cancers.
  • References (3)

    3.1 Defining CD 8+ T cell responses against Bax pool 1-15 100 3.1.1 Peptide binding to HLA-A2 on the T2 cell line 100 3.1.2 D onor responses 102 3.2 G eneration of a Bax-specific polyclonal T cell line using CD107 assay 106 3.2.1 M apping epitopes from Bax pool 1-15 responses using the JSBline 108 3.2.2 Loss of Bax peptide specificity through repeated expansions of the JSB line 110 3.3 Defining CD8+ T cell responses against Bax pool 601-23 112 3.3.1 Peptide binding to HLA-A2 on the T2 cell line 114 3.3.2 D onor responses 116 3.4 Testing different protocols for the detection of peptide specific T cells 118 3.5 G eneration of T cell lines against Bax pool 601-23 using IFNy secretion assay 121 3.5.1 Enrichm ent of IFN y-secreting cells from donor 11 using m agnetic-based 121 cell sorting 3.5.2 G eneration of KSI line through the antigen-independent expansion 124 of IFNy-enriched T cells 3.5.3 G eneration of Bax-specific KSI 10B7 T cells using the lim iting dilution 124 protocol

    3.6. G eneration of T celllines from JSB cells using IFNy secretion assay 126 3.6.1 Enrichm ent of IFNy-secreting cells from JSB cells using m agnetic-based 126 cell sorting 3.6.2 G eneration of JSBI line through the antigen-independent expansion 128 3.6.3 G eneration of Bax-specific JSBI 10B6 T cells using the lim iting dilution 128 protocol

    3.7 D iscussion 130 Boslego J, Sattler C, Barr E, Koutsky LA (2007) Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. N Engl J M ed 356(19): 1928-1943

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