Page 292 - Haematologica March 2020

M. Ando et al.
challenging. Bollard and colleagues successfully demon- strated effective control EBV-associated lymphomas with either type II or type III latency using patients’ own LMP1- and LMP2-specific T cells: among six NK/T cell lymphoma (EBV-infected) patients whose tumors had relapsed after standard chemotherapies and who had received LMP1 or LMP2-specific CTL, three remained in CR, with responses that were associated with percent- ages of effector and central memory LMP1-specific T cells in the infused population.16 Functionally rejT differentiat- ed from T-iPSC include younger phenotypes such as cen- tral memory and effector memory phenotypes and have longer telomeres and a stronger proliferation ability (100- fold to 1,000-fold after T-cell stimulation) than original peripheral-blood derived CTL.18-20,30 Although the original CTL expressed PD-1 strongly, their redifferentiated LMP2-rejT descendants almost lacked PD-1 expression (Figure 3C). CTL generation from heavily treated patients is generally more difficult than from healthy donors because of T-cell exhaustion. Although CTL clones from an ENKL patient showed relatively strong cytotoxicity against EBV-infected autologous LCLs in vitro (Figure 3A), the proliferation ability of patient-derived CTL clones was lower than that of healthy donor-derived CTL clones, and much lower than that of rejT. LMP2-rejT had a distinct survival advantage in ENKL-bearing mice over original LMP2-CTL (Figure 4C). Histopathological exami- nation revealed that LMP2-rejT completely eradicated ENKL and that LMP2-rejT persisted in the spleen of long- surviving ENKL-bearing mice, supporting our hypothesis that rejT contribute to ENKL eradication as long-lived memory T cells (Figure 5A). Furthermore, we actually confirmed the presence of central memory phenotype human T cells in the peripheral blood of a long-surviving ENKL-bearing LMP2-rejT treated mouse (Figure 5B). However, neither necrotic lesions nor features of activa- tion of immune cells (which might lead to organ injury) were found in these long-surviving mice, suggesting that long-term persistence of rejT does not reduce safety in this in vivo model.
As EBV-associated lymphomas express PD-L1 more strongly than lymphomas without EBV infection,23-26 we examined PD-L1 expression in ENKL cells. Our statistical analysis demonstrated that PD-L1 expression was clearly related to very poor prognosis in ENKL patients (Figure 2A). Therefore, we anticipated that blockade of PD-1 and PD-L1 engagement would reinforce the efficacy of treat- ments using EBV-specific CTL expressing PD-1 and possi- bly EBV-rejT not expressing PD-1 for PD-L1 - expressing ENKL. Contrary to our expectations, we could not observe clear treatment enhancement by anti-PD-1 Ab with either original EBV-CTL or EBV-rejT (Figure 4 B-C), suggesting that PD-1 blockade is not necessarily required in treatment of ENKL with EBV-specific T cells. Anti-PD- 1 Ab reportedly had no measurable effect on chimeric antigen receptor (CAR) T-cell expansion, persistence, or circulating cytokine levels when it was administered in combination with CAR T cells and lymphodepletion.31 In our study, EBV-rejT alone effectively ablated ENKL in tumor-bearing mice, with such strong anti-tumor effects
that any additive beneficial effects of anti-PD-1 Ab were unclear. Of relevance is that toxicities of anti-PD-1 Ab may have impaired survival: disruption of the PD-1/PD-L1 pathway can lead to imbalances in immunologic toler- ance, resulting in unchecked autoimmune-like/inflamma- tory side effects.32 By contrast, viral-specific antigen-spe- cific CTL therapy is minimally toxic and does not harm healthy tissues.16 We postulate that also EBV-rejT therapy is free from severe adverse events and suggest that it will be highly effective against ENKL when L-asparaginase therapy has failed.
EBV-rejT still exerted strong cytotoxic effects against tumor cells in ascites from mice with relapse after EBV- rejT therapy. These tumor cells maintained HLA class I expression and harbored no LMP2 mutations (Figure 6). This suggests that EBV-rejT may not fully penetrate all areas of tissue metastasis. We did not administer cytokines such as IL-2, IL-7 and IL-15 to mice to avoid an artificial increase of the activity of ENKL cells in vivo, because even without cytokines ENKL cells rapidly prolif- erated in mice and the tumor signal progressively increased. Using cytokines, the expansion of rejT might be much stronger and the incidence of relapse might decrease. However, it is encouraging that even without cytokines, rejT were well activated by recognizing ENKL cells and showed strong cytotoxic activity against ENKL cells.
Our results collectively suggest that to treat relapsed and refractory ENKL using LMP1- and LMP2-specific rejT will be very useful, as large numbers of functionally rejuvenat- ed LMP1- and LMP2-specific CTL can reliably be obtained from T-iPSC. The greatest advantage of rejT therapy is that once T-iPSC are established from an EBV-CTL clone, therapeutic T cells can be generated from T-iPSC in unlim- ited numbers. If patient tumor cells strongly express PD- L1, the associated poor prognosis should prompt care- givers to generate patient-specific EBV-rejT targeting LMP1 and LMP2 while SMILE therapy is administered. EBV-rejT can supply a reliable salvage therapy for refrac- tory and relapsed ENKL in which L-asparaginase therapy has failed. To establish banks of T-iPSC directed against a variety of viral antigens and HLA types may ultimately provide effective “off-the-shelf” T-cell adoptive immunotherapy treatments against EKNL. The principle demonstrated here can be extended to other virus-induced tumors and neoantigens.
Acknowledgments
We thank A.S. Knisely for critical reading of the manuscript; Kazuo Ohara, Tokuko Toyota and Masako Fujita for technical help with cell culture; Azusa Fujita and Yumiko Ishii for FACS operation; Gianpietro Dotti and Nobuhiro Nishio provided retro- viral FFluc-GFP plasmid; Mahito Nakanishi and Manami Otaka provided Sendai virus vector. We also thank Motoo Watanabe, Hajime Yasuda, and Kazuo Oshimi for helpful dis- cussions. The project was supported by JSPS KAKENHI Grant Number 15J40133 and 16K09842. The institutional regulation boards for human ethics at Juntendo University School of Medicine and at the Institute of Medical Science, University of Tokyo, approved the experimental protocol.
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