Establishment of a stable T lymphoma cell line transduced with HLA-A^|^lowast;24:02-restricted WT1-specific TCR genes and its application to antigen-specific immunomonitoring

Abstract

Wilms' tumor gene 1 (WT1) has been proposed as an attractive target for cancer immunotherapy. A natural 9-mer peptide (CYTWNQMNL), which bound to human leukocyte antigen (HLA)-A*24:02, was identified from among WT1-specific cytotoxic T lymphocyte (CTL) epitopes. This natural WT1 CTL epitope peptide was further modified (CMTWNQMNL) to enhance its binding affinity to HLA-A*24:02. This modified WT1 CTL epitope peptide was superior to the natural peptide for inducing HLA-A*24:02-restricted WT1-specific CTLs. Here we induced several WT1 CTLs that reacted with both modified and natural WT1 tetramers from peripheral blood mononuclear cells. Then, T-cell receptor (TCR) genes were isolated from these WT1 CTLs to determine their Vα and Vβ usage. These TCR genes were transduced into human T lymphoma cells to establish a stable cell line, SK37, which expressed a WT1-specific TCR. We confirmed that SK37 cells reacted with both modified and natural WT1 tetramers, which indicated that SK37 cells could be a useful tool for WT1 tetramer reagent quality assurance. One the basis of these findings, we propose that this WT1 tetramer, which was quality-assured using established SK37 cells, will contribute to reliable immunomonitoring of tumor-specific CTL responses of cancer patients who receive WT1-targeted cancer vaccine therapy or TCR-gene therapy.

Identification of TCR Vβ11-2-Dβ1-Jβ1-1 T cell clone specific for WT1 peptides using high-throughput TCRβ gene sequencing

“…The reason that different TCR clone amplification induced by the same peptide, is due to the individual T cell response from different donors and patients. There were also studies showing different TCR clone ( TCR Vβ 5-1- Dβ 2- Jβ 2-5) inducted by WT1 (CMTWNQMNL) peptides [44]. Overall, the new identified TCR V β 11-2- Dβ 1- Jβ 1-1 clone in this study may provide new data of WT1 specific TCR clone bank.…”

Development of a T‐cell receptor multimer with high avidity for detecting a naturally presented tumor‐associated antigen on osteosarcoma cells

“…To investigate CD8 dependency of TCR‐HLA interaction, ITG‐MT3 TCR and FKS‐D11P TCR genes were transfected to J.RT3‐T3.5 cells, CD8 and TCR β chain‐deficient mutant Jurkat cells . As a result, both transduced TCR could react with specific tetramers, but the CD8‐dependent WT1 TCR could not react with the WT1 tetramer (Figure A). These results suggested that both ITG‐MT3 and FKS‐D11P TCR could react with the peptide/HLA complex in a CD8‐independent way.…”

“…Affinity of FKS-D11P TCR (K D value of 4.70 × 10 −9 mol/L) was much higher than that of naturally occurring TCR binding to HLA class I/peptides (K D value of 1-50 × 10 −6 mol/L). 21 In previous studies on a soluble TCR-multimer, K D values of TCR for HLA-A2/HTLV type 1 Tax [11][12][13][14][15][16][17][18][19] , HLA-A2/HBV env [183][184][185][186][187][188][189][190][191] , HLA-A2/MART1 26-35(A27L) and HL A-A2/NY-ESO-1 157-167(C165V) were 1.06 × 10 −6 , 0.6 × 10 −6 , 5.4 × 10 −6 and 4.3 × 10 −6 mol/L, respectively. 17,19,20 These results suggested that the affinities of successfully constructed soluble TCR were also higher than the general affinity of TCR.…”

“…The TCR β chain is composed of a TCR beta chain variable region (TRBV), a diversity region (TRBD), a joining region (TRBJ), and a constant region (TRBC). Cloning and sequencing of TCR α/β genes using PCR were carried out as previously described . Briefly, total RNA from sorted CTL was prepared with an RNeasy Mini Kit (QIAGEN, Hilden, Germany), and an aliquot (1 μg) was subjected to reverse transcription using an oligo (dT) primer and SuperScript III (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Cloning and sequencing of TCR α/β genes using PCR were carried out as previously described. 11 Briefly, total RNA from sorted CTL was prepared with an RNeasy Mini Kit (QIAGEN, Hilden, Germany), and an aliquot (1 μg) was subjected to reverse transcription using an oligo (dT) primer and SuperScript III (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was amplified by PCR with coding region-specific primers for TRBV12-3/4 and TRBC1/2 (TCR β gene of ITG-MT3), TRBV9 and TRBC1/2 (TCR β gene of FKS-D11P), and various TRAV primers and TRAC (TCR α chain).…”