Seran C. Hill

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

In this study, we describe the expression and function of CD40, a TNF receptor family member, in cervical carcinomas. CD40 was present at very low levels in normal cervical epithelium but was overexpressed in human papillomavirus-infected lesions and advanced squamous carcinomas of the cervix. The stimulation of CD40-positive cervical carcinoma cell lines with soluble CD40L (CD154) resulted in activation of the NF-kappaB and MAPK signaling pathways and up-regulation of cell surface markers and intracellular molecules associated with Ag processing and presentation. Concomitantly, the CD154-induced activation of CD40 in carcinoma cells was found to directly influence susceptibility to CTL-mediated killing. Thus, CD40 stimulation in cervical carcinoma cell lines expressing a TAP-dependent human papillomavirus 16 E6 Ag epitope resulted in their enhanced killing by specific CTLs. However, CD154 treatment of carcinoma cells expressing proteasome-dependent but TAP-independent Ags from the EBV-encoded BRLF1 and BMLF1 failed to increase tumor cell lysis by specific CTLs. Moreover, we demonstrate that chemotherapeutic agents that suppress protein synthesis and reverse the CD40-mediated dissociation of the translational repressor eukaryotic initiation factor 4E-binding protein from the initiation factor eukaryotic initiation factor 4E, such as 5-fluorouracil, etoposide, and quercetin, dramatically increase the susceptibility of cervical carcinoma cells to CD40L-induced apoptosis. Taken together, these observations demonstrate the functional expression of CD40 in epithelial tumors of the cervix and support the clinical exploitation of the CD40 pathway for the treatment of cervical cancer through its multiple effects on tumor cell growth, apoptosis, and immune recognition.

The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.

Cytotoxic T lymphocytes are an essential component of the anti-tumour immune response and they are activated by binding of the T cell receptor to a peptide-MHC complex on the tumour cell surface. These surface-bound peptides originate from intracellular proteins that have been degraded into peptides by the proteasome and presented by MHC class I.
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\nMany tumours inappropriately express mutated or overexpressed self-proteins (p53, ras), tissue specific antigens (MAGE, MelanA) or tumour-specific fusion proteins (bcr-abl) which, due to their tumour association can be used as targets for anti-tumour immunotherapy.
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\nThis study presents a technique to isolate and identify surface-MHC bound tumour antigen peptides from tumour cells. Peptide isolation was achieved using a mild acid buffer to destabilise surface MHC class I molecules; concentration and purification of the peptides was facilitated by ion-exchange-HPLC and subsequent peptide sequence information was generated by electrospray mass spectrometry. The sequence data was then entered into a database to determine the protein origin.
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\nPeptides were isolated from an HLA-A3 transfected K562 cell line model, which naturally expresses bcr-abl but no other HLA class I molecules, to determine whether peptides from the fusion region were naturally presented by HLA-A3. PBMC from HLA- A3 positive CML patients was then analysed in the same way. The identification of bcr-abl fusion region peptides from the K562 transfectants and HLA-A3 positive CML patient material is, to the best of my knowledge, the first conclusive demonstration that the bcr-abl fusion protein is processed intracellularly resulting in the surface expression of peptides from the fusion region. These peptides represent an entirely tumour specific antigen and there is potential for their use as a target in anti-tumour immunotherapeutic strategies.