WT1-specific cytotoxic T lymphocytes induced by activated B cells as apc

Abstract

Background & Aim WT1 is highly expressed in many malignancies including leukemia and targeting WT1 as a Tumor Associated Antigen (TAA) in cancer immunotherapy is attractive. In this study, we induced WT1-specific cytotoxic T lymphocytes to verify that activated B cells can act as cancer antigen presenting cell and induce CTLs. Methods, Results & Conclusion For the generation of CTLs against WT1, activated B cells were used as antigen presenting cells. B cells were isolated from PBMCs of several normal healthy donors and activated with α-galactosylceramide (α-GalCer) and nucleofected with target antigen-coding plasmid DNA with methods based on the previous report (Cytotherapy. 2017;19:119-127). Activated B cells were then cultured with PBMCs for 17 days in vitro and harvested for assay. Cells expanded about 3 times after 17 days of culture. We examined characteristic of WT1-specific CTLs by their surface markers. WT1-specific CTLs had more than 90% CD3+, and ratio of CD8 to CD4 was 1.7-5.8. The CTLs showed a decrease in naive cells (CD62L+CD45RA+) and an increase in effector memory cells (CD62L+CD45RA-) and central memory cells (CD62L-CD45RA-) compared with non-stimulated PBMCs. Subsequently, the IFN-γ ELISPOT (Enzyme-linked immunospot) assay was performed to confirm the response of the induced WT1-specific CTLs to the WT1 antigen. When WT1-specific CTLs encounters target that does not have a WT1 antigen, they did not produce IFN-γ, but when they encounters target cells loaded with WT1 antigen, it responded to secrete IFN-γ. Killing assay was also performed to determine the immunogenicity of induced CTLs. The induced WT1 CTLs were found to kill more than 90% of autologous PBMCs loaded with WT1 pepmix when the E:T ratio was 10:1. In addition, we found that WT1 CTLs have killing activity when they encounter leukemia cell lines that express WT1 and HLA-A*0201 matched. We can induce WT1-specific CTLs and this study confirmed that B cells activated by α-GalCer can act as WT1 presenting cells to induce WT1 specific CTLs. Our results indicate that these in vitro expanded WT1-specific CTLs using activated B cells can be a candidate for adoptive immunotherapy against cancer.