Efficient Generation of HLA-A24-Restricted WT1-Specific Cytotoxic T Lymphocytes Using Gene-Engineered Artificial Antigen-Presenting Cells

Recent success associated with adoptive transfer of antitumor T cells in lymphodepleted patients suggests the potential of adoptive immunotherapy to have a significant clinical impact. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we previously reported a culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL) from HLA-A2-positive melanoma patients by using K562-based artificial antigen-presenting cells (aAPCs). In the present study, we have applied the culture system to HLA-A24, one of the most common HLA class I antigen in the Asian population, and examined whether HLA-A2402-restricted WT1-specific CTL can be generated using aAPCs.

HLA-A2402–positive peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (n=4) and cancer patients (n=10). To establish antigen-specific T cells, CD8⁺ T cells were purified by positive selection using a magnetic beads method (Miltenyi Biotec). aAPCs were pulsed with an HLA-A2402 restricted, modified 9-mer WT1 immunodominant peptide (CYTWNQMNL). aAPCs were then irradiated with 200 Gy and added to purified CD8⁺ T cells at a ratio of 1:20 in 96-well plates in RPMI1640 supplemented with 10% human AB serum. Between stimulations, IL-2 (10 U/ml) and IL-15 (10ng/ml) (both from Peprotech) were added to the cultures.

Flow cytometry (FACS) analysis confirmed that aAPCs stably expressed transduced HLA class I, CD80 and CD83 molecules. aAPC did not express HLA class II molecules, CD40, CD154, or CD86. Following 3–4 rounds of weekly stimulation with peptide-pulsed aAPCs, WT1 peptide-specific CD8⁺ T cells were evaluated by a tetramer staining. The percentage of tetramer-positive cells was 0.082±0.0075% before stimulation. It increased to 0.865±0.528% (10.5 fold increase) and 1.89±1.12% (23.0 fold increase) following the third and the forth stimulation, respectively. There was no marked difference in magnitude of increase between healthy donors and cancer patients. However, when CD8⁺ T cells from patients vaccinated with WT1-peptide pulsed dendritic cells were stimulated with WT1-peptide-pulsed aAPCs, the percentage of tetramer-positive cells was significantly higher (11.72±6.46%) following the third stimulation. CD8⁺ T cells stimulated with WT1-peptide-pulsed aAPCs were negative for both A0201/WT1 and A2402/CMV tetramers, confirming HLA restriction and antigen specificity. FACS analysis revealed that WT1-specific CTL expanded with WT1-peptide-pulsed aAPCs expressed a memory phenotype. Furthermore, these CTL demonstrated cytotoxicity against CIR-A2402 target cells pulsed with a WT1 peptide in an LDH release assay. Unpulsed or HIV peptide-pulsed target cells were not lysed.

These results demonstrated that HLA-A24-restricted WT1 specific CTLs with a memory phenotype can be generated ex vivo using peptide-pulsed gene-engineered aAPCs within a short period of time for clinical use.

No relevant conflicts of interest to declare.