Enzyme-Linked Immunosorbent Spot Assay for the Detection of Wilms’ Tumor 1-Specific T Cells Induced by Dendritic Cell Vaccination

Abstract

Background: Despite recent advances in cancer immunotherapy and the development of various assays for T cell assessment, a lack of universal standards within immune monitoring remains. The objective of this study was to evaluate the enzyme-linked immunosorbent spot (ELISpot) assay in comparison with major histocompatibility complex-tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. Methods: The ELISpot assay was performed on peripheral blood mononuclear cells to assess reproducibility, daily precision, and linearity using 305HLA-A*24:02-restricted Cytomegalovirus peptide. Wilms' tumor 1 (WT1) antigen-specific cytotoxic T cells were then evaluated by both the ELISpot assay and WT1 tetramer analysis in peripheral blood from 46 cancer patients who received DC vaccinations pulsed with human leukocyte antigen (HLA)-A*24:02-restricted modified WT1 peptides. Results: The ELISpot assay was proven to have reproducibility (coefficient of variation (CV) ranged from 7.4% to 16.3%), daily precision (CV ranged from 5.0% to 17.3%), and linearity (r = 0.96-0.98). WT1-specific immune responses were detected by the ELISpot assay in 34 out of 46 patients (73.9%) post-vaccination. A Spearman's rank-correlation coefficient of 0.82 between the ELISpot assay and WT1 tetramer analysis was obtained. Conclusion: This is the first report of a comparison of an ELISpot assay and tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. The ELISpot assay has reproducibility, linearity, and excellent correlation with the WT1 tetramer analysis. These findings suggest that the validated ELISpot assay is useful to monitor the acquired immunity by DC vaccination targeting WT1.

Updates on immunotherapy for colorectal cancer

“…Other TAAs have also been utilized in DC vaccines as demonstrated by a recent phase I trial evaluating the safety and immunogenicity of Wilms' tumor (WT1) class I/II peptides based DC vaccine for patients with advanced CRC (41). This trial confirmed DC vaccine efficacy based on WT1 expression in tissue using immunohistochemistry and identification of WT1-specific cytotoxic T cells using the Enzyme-Linked Immunosorbent Spot (ELISPOT) assays (Mabtech, Nacka Strand, Sweden) (42). Interestingly, the DC vaccine immunity persisted for two years associated with a prolongation in survival.…”

Dendritic Cells Pre-Pulsed with Wilms’ Tumor 1 in Optimized Culture for Cancer Vaccination

“…Dead cells were excluded via 7-AAD (BD Pharmingen) staining for flow cytometry. WT1 tetramer-positive CTLs were defined according to the following criteria: (1) comprising at least 0.02% of the CD3 + CD8 + subset of 50,000-100,000 lymphocytes and (2) forming a clustered but not diffuse population [38].…”

An update on Dendritic Cell-Based Cancer Immunotherapy

“…The presence of WT1 antigen-specific cytotoxic T cells (WT1-CTLs) is defined according to the following criteria: presence of greater than 0.02% WT1-positive CD8 + T cells among the 50,000-10,000 lymphocytes analyzed with no evidence of false positive cells and WT1-positive cell population being clustered but not diffused with slight modification, as previously described [64]. ELISPOT assays were performed to measure WT1-specific IFN-γ production by peripheral blood mononuclear cells (PBMCs).…”

“…ELISPOT assays were performed to measure WT1-specific IFN-γ production by peripheral blood mononuclear cells (PBMCs). The presence of WT-CTLs was defined according to the following criteria: presence of at least 15 WT1-specific spots per 1 × 106 PBMCs and at least 50% more WT1-specific spots than negative peptide (HIV peptide) spots [64].…”