Redirected CD4+ T Cells Using WT1-Specific T-Cell Receptor Gene Transfer Can Supply Multifactorial Help to Enhance the Anti-Leukemia Reactivity Mediated by Similarly Redirected CD8+ T Cells Using the Identical Gene Transfer
Authors: Ochi Toshiki , Fujiwara Hiroshi , Okamoto Sachiko , Asai Hiroaki , Miyazaki Yukihiro , Shirakata Toshiaki , Mineno Junichi , Kuzushima Kiyotaka , Shiku Hiroshi , Yasukawa Masaki 
Published: October 2019

Abstract
Abstract 645
[Purpose] Redirected T-cell-based anti-cancer adoptive therapy using cancer antigen-specific T-cell receptor (TCR) gene transfer has clinically shown promise, however there still remain considerable issues of achieving better clinical efficacy. For this purpose, helper function provided by concurrent CD4+ T cells should be evidently considerable. In this study, to achieve the enhanced anti-leukemia functionality mediated by redirected CD8+ T cells using WT1-specific TCR gene transfer, we in detail examined functionalities mediated by similarly redirected CD4+ T cells using the identical TCR gene transfer.[Methods] HLA-A*24:02-restricted and WT1235-243-specific codon-optimized TCR α/β genes were inserted into the novel retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector). (1) Cognate antigen-responsive cytokine production mediated by WT1-siTCR transduced CD4+ T cells was assessed using cytokine beads array and ELISA assay. (2) Expression of CD40L and OX40 on redirected CD4+ T cells stimulated by WT1 peptide ligation was assessed using flow cytometer. (3) Impact caused by redirected CD4+ T cells on each magnitude of WT1-specific cytotoxicity, target-responsive proliferation and transition to central memory T-cell phenotype of WT1-siTCR transduced CD8+ T cells was measured using 51Cr-release assay, CD107a assay, intracellular IFN-γ assay and CFSE assay. (4) Chemokines produced by redirected CD4+ T cells stimulated using WT1 peptide was comprehensively assessed using real-time PCR. Consequent chemotaxis of redirected CD8+ T cells toward those stimulated redirected CD4+ cells was validated using transwell experiments. (6) Finally, anti-leukemia reactivity against autologous leukemia cells mediated by patients' redirected CD8+ T cells was similarly examined in the presence or absence of such autologous CD4+ T cells.

[Results]
First, in this study, those redirected CD4+ T cells hardly became positive for intracellular FoxP3, a crucial marker for regulatory T cell phenotype. In response to the WT1 peptide, WT1-siTCR transduced CD4+ T cells produced Th1 cytokines; IL-2, IFN-γ and TNF-α, in the context of HLA-A*24:02, which also needed HLA class II molecules on target cells. Magnitudes of WT1-responsive CD107a expression, IFN-γ production and cytotoxicity mediated by WT1-siTCR transduced CD8+ T cells were efficiently enhanced in the presence of redirected, but not non-redirected CD4+ T cells. Similarly, in the presence of those redirected CD4+ T cells, redirected CD8+ T cells expressing WT1-specific TCR increased in number and the transition to central memory T-cell phenotype (CD45RA−CD62L+) of those CD8+ T cells was stimulated in response to the stimulation with WT1 peptide. WT1 peptide ligation stimulated those redirected CD4+ T cells to express membrane-bound OX40, which is involved in the formation of central memory CD8+ T cells. WT1 peptide ligation also stimulated the redirected CD4+ T cells to produce chemokines; CCL3 and CCL4. Redirected CD8+ T cells expressed the receptor for these chemokines, CCR5; efficient migration of redirected CD8+ T cells toward redirected CD4+ T cells stimulated with WT1 peptide was obviously observed in transwell experiments. Finally, redirected CD4+ T cells isolated from patients with leukemia successfully provided Th1 helper function to autologous redirected CD8+ T cells to enhance the anti-leukemia reactivity; cytotoxicity, proliferation and formation of central memory T cells, in response to autologous leukemia cells, in vitro.

[Conclusion]
Although further investigations are warranted, concurrently adopted WT1-siTCR introduced CD4+ T cells seems feasible to enhance the efficacy of WT1-targeting redirected T-cell-based adoptive therapy for treatment of human leukemia.

Disclosures:
No relevant conflicts of interest to declare.