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Long-term eradication of ENKL by rejuvenated CTL
days, by contrast, contained many fewer human CD3+ T cells (Figure 5A, lower row of panels). Flow cytometry also was used to detect LMP2-rejT in the peripheral blood of ENKL-bearing mice treated using LMP2-rejT and sur- viving long-term. LMP2-tetramer – expressing CD3+ human T cells were present. Among them were effector memory (CD45RA–, CD62L–) and central memory pheno- type T cells (CD45RA–, CD62L+ , CD27+ , CD95+ ) (Figure 5B).
Four of six mice that received rejT injections survived 182 to 296 days after treatment. Two relapsed, developing ascites before day 100. Tumor cells in ascitic fluid retained HLA class I expression (Figure 6A) without mutation in LMP2 (Figure 6B). Resistance to LMP2-rejT therapy in ascitic-fluid tumor cells was evaluated by 51Cr release assay. EBV-rejT specific for LMP2 showed robust cytotox- icity (77%, 71.6%, 67.1%, and 51.2%; E:T ratios of 40:1, 20:1, 10:1 and 5:1 specific 51Cr release, respectively) against ENKL cells in ascites (Figure 6C). These findings indicated that mutant ENKL cells did not appear after three rejT treatments and that the tumor cells had not developed resistance to LMP2-rejT therapy.
A
Discussion
We sought to verify that iPSC-derived rejT therapy spe- cific to LMP1/LMP2 antigen would be an effective salvage therapy for refractory and relapsed ENKL. Although L- asparaginase is a key drug for ENKL4 and L-asparaginase- containing chemotherapies improve prognosis in advanced ENKL patients,5,6 tumor in about half of these patients resists L-asparaginase therapy or relapses after remission, resulting in a miserable clinical course. Adoptive T-cell therapy using peripheral blood-derived EBV-specific CTL is clearly effective for EBV-driven lym- phoproliferative diseases after hematopoietic stem cell transplantation.11-14 As these lymphomas develop in immunosuppressive situations and show type III latency, EBV-specific CTL adjuvant therapy can induce durable remissions. However, EBV-associated lymphomas show- ing type II latency, such as ENKL, Hodgkin lymphoma, and diffuse large B-cell lymphomas, are weakly immuno- genic, with lymphoma cells that express only LMP1 and LMP2 antigens.15 Therefore, EBV-CTL therapy targeting EBV-associated lymphomas with type II latency is more
Figure 6. Phenotype of extranodal NK/T-cell lymphoma, nasal type cells in ascites of relapsed mice treated with LMP2-rejT. (A) Flow cytometric analysis of HLA class I (A, B, C) expression on NK- YS cells in ascites. (B) LMP2 sequence of post-rejT NK-YS cells in ascites. (C) In vitro 51Cr release assay of EBV-rejT (effector), and HLA mismatched T cells (control effector) against NK-YS cells in culture and NK-YS cells in ascites (targets). Data are presented as mean ± SD. E:T ratio: effector : target ratio; ENKL: extranodal NK/T-cell lymphoma, nasal type; LMP: latent membrane protein; HLA: human leukocyte antigen; EBV: Epstein-Barr virus; SD: stan- dard deviation.
B
C
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