Dominant responses with conservation of T-cell receptor usage in the CD8+ T-cell recognition of a cancer testis antigen peptide presented through HLA-Cw7 in patients with multiple myeloma

Abstract

Cancer testis antigens exhibit physiological expression within germ cells and are frequently expressed in malignant tissue. Interestingly, immunological tolerance to cancer testis proteins does not appear to be established, and the expression of CTAg proteins within malignant cells can therefore lead to induction of cellular and humoral immunity. A considerable body of evidence now indicates that CD8-specific immunity plays an important role in the control of cancer cell growth, and a number of vaccine studies are in progress to boost CTAg-specific cellular immune responses. We have previously identified CTAg-specific immune responses in patients with multiple myeloma and reported that recognition of the MAGE-A1(289-298) peptide, which is described as being restricted by HLA-B*0702, was the most frequent response seen with our peptide panel. Here, we studied seven CD8+ T-cell clones specific for this peptide which were isolated from three patients with myeloma at several time-points. The affinity of peptide recognition was high with 50% maximal interferon-γ production observed at a peptide concentration of 10(-10) M and variation of only one order of magnitude between the affinities of the clones. Importantly, all the clones were able to recognise and kill multiple myeloma cell lines. Interestingly, one patient did not express HLA-B*0702, but three clones from this patient recognised the MAGE-A1(289-298) peptide on a lymphoblastoid cell line (LCLs) expressing HLA-Cw7, and we now show evidence that the MAGE-A1(289-298) peptide is expressed and recognised through Cw7. The T-cell receptor gene usage was determined in five clones and showed conserved features in both the α and the β chain genes indicating correlation between T-cell receptor usage and peptide specificity of cancer testis antigen-specific T-cell clones.

Fig. 1

The specificity and magnitude of the CD8+ T-cell response to CTAg peptides in patients with multiple myeloma. a Individual peptide responses were calculated through use of the IFN-γ secretion assay (Miltenyi Biotec). The range of CTAg peptides is shown on the x-axis, and the maximal CTAg-specific T-cell response from each patient is shown on the y-axis and represented as a proportion of the total CD8+ T-cell pool. b Comparison of the frequency of the MAGE-A1_289–298 response compared with the other detectable CTAg-specific CD8+ T-cell responses. Mann–Whitney T test was used to calculate the P value (0.0082)

Fig. 2

Functional analysis of MAGE-A1_289–298-specific CD8+ T-cell clones generated from MM patients 4 and 17. a, b IFN-γ production and cytotoxicity properties of generated MAGE-A1_289–298 (RVRF) CD8+ T-cell clones. a, b (i) MAGE-A1_289–298 (RVRF)-specific CD8+ T cells were stimulated with peptide-loaded or control peptide-loaded HLA-matched or mismatched LCL target cells or MM cell lines U266, JJN3 and H929. a (ii) Cytolytic activity of the CD8+ T cell clones by standard 6-h chromium release assay at an effector-target ratio of 10:1 against peptide or control peptide-loaded HLA-matched target cells or MM cell lines

Fig. 3

Recognition of a peptide-loaded LCL that is HLA B7−, Cw7+ by MAGE-A1_289–298-specific CD8+ T-cell clones generated from patient 4. IFN-γ production of the MAGE-A1_289–298 (RVRF)-specific CD8+ T-cell clone 22 generated from patient 4 was assessed using IFN-γ ELISA. The clone was stimulated with peptide-loaded or control peptide-loaded HLA-defined LCL target cells or multiple myeloma cell lines U266, JJN3 and H929 or K562 (NK-sensitive cell line)

Fig. 4

Peptide titration analysis of CD8+ T-cell clones from MM patients 4 and 17. a, b The affinity of the T-cell clones is represented as a percentage of IFN-γ release with lines representing the peptide concentration required for clones to generate 50% of maximal IFN-γ release