Abstract
In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126–188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20–23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides.
Highlights
For many years the measurements of the levels of cellular immune responses, e.g., those mediated by T cells, depended largely on in vitro culture and subsequent measurement of specific functions like cytotoxicity, proliferation or bulk cytokine production
Among the different methods available for monitoring of CD8+ T cells responses due to its high throughput, sensitivity and robustness the ELISPOT assay represents the method of choice in many laboratories
In the present study we examined the capability of using longer peptides when scrutinizing PMBC from melanoma patients for spontaneous immunity by means of ELISPOT IFN- secretion assay
Summary
For many years the measurements of the levels of cellular immune responses, e.g., those mediated by T cells, depended largely on in vitro culture and subsequent measurement of specific functions like cytotoxicity, proliferation or bulk cytokine production. New approaches to monitor and analyze anti-tumor immune responses, requiring minimal in vitro manipulations have opened new avenues to characterize spontaneous as well as treatment-induced T-cell responses [1]. To this end, technical advantages allow the detection of low frequencies of precursor CD8+ T cells with high sensitivity. Among the different methods available for monitoring of CD8+ T cells responses due to its high throughput, sensitivity and robustness the ELISPOT assay represents the method of choice in many laboratories. The ELISPOT assay is based on the detection of antigen-induced release of cytokines—most often IFN-—by single T cells upon triggering of its TCR [2].
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