Page 283 - Haematologica March 2020

Long-term eradication of ENKL by rejuvenated CTL
longer telomeres than the original CTL, iPSC-derived CTL are functionally rejuvenated T cells (rejT).17 The rejT that we generated have strong anti-tumor effects against EBV- infected lymphoblastoid cells (LCL) in vivo and in mice they confer a survival advantage compared to mice treated using original CTL.19 Hence, rejT therapy directed against LMP1 and LMP2 is expected to be useful as a salvage ther- apy for ENKL in which SMILE has failed.
Another factor in ENKL prognosis is the PD-1 pathway for immunoevasion.21-23 EBV-associated lymphoma cells often express the PD-1 ligand (PD-L1).24-26 The complete remission (CR) rate is high after PD-1 blockade with pem- brolizumab in ENKL in which L-asparaginase therapy has failed.27
We sought to demonstrate the effectiveness of rejT ther- apy targeting ENKL. We also investigated additive anti- tumor effects of PD-1 blockade in conjunction with rejT therapy. Both rejT and original CTL showed robust tumor-suppressive effects against ENKL in vitro and in vivo. However, only LMP2-specific rejT significantly prolonged long-term survival in ENKL-bearing mice. This effect was so strong that any additive anti-tumor effect of PD-1 blockade was overwhelmed. LMP1- and LMP2-specific rejT therapy seems a promising salvage therapy for ENKL refractory to SMILE.
Methods
More detailed information can be found in the Online Supplementary Data.
Patients and samples
We reviewed 28 biopsy samples from 24 patients diagnosed with ENKL at the Juntendo University School of Medicine, Department of Hematology, between 2006 and 2017. The use of the material and clinical information was approved by the Research Ethics Committee for the Faculty of Medicine, Juntendo University, and was in accordance with the Declaration of Helsinki.
Immunohistochemical staining
Tissue samples were fixed in formalin and embedded in paraf- fin. Anti-PD-L1 rabbit monoclonal antibodies (EPR1161[2]; 1:200 dilution; ab174838, Abcam, Cambridge, MA), anti-PD-1 mouse monoclonal antibodies (NAT105; 1:100 dilution; ab52587, Abcam), and anti-CD3 rabbit monoclonal antibodies (SP7; 1:50 dilution; ab16669, Abcam) were used for immunostaining.
Generation of LMP1/2-specific CTL and establishment of T-iPSC
LMP1/2-specific CTL were generated using peripheral blood mononuclear cells (PBMC) obtained from two human leukocyte antigen (HLA)-A*2402-expressing healthy donors and one HLA- A*0201-expressing ENKL patient. Selected clones were transduced with Sendai virus vectors to establish T-iPSC.
T-cell differentiation from T-iPSC
To differentiate human iPSC into hematopoietic cells, small clumps of iPSC were transferred onto C3H10T1/2 cells. Hematopoietic cells collected from iPSC sac contents were trans- ferred onto DL1/4-expressing C3H10T1/2 feeder cells. T-lineage cells were then harvested and stimulated. The antigen specificity of LMP1/2-specific rejT was determined by staining with LMP1/2 tetramer.
Cell lines
ENKL cells (NK-YS and SNK-6 lines) were grown in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of interleukin-2 (IL-2) and in NS-A2 (GreenDay) supplemented with 100 U/mL of IL2, respec- tively.4,28
51Cr release assays
Cytotoxic specificity of EBV-CTL and EBV-rejT directed at LMP1/2 antigen was analyzed in a standard 4-hour 51Cr- release assay at different effector : target ratios (E:T; 40:1, 20:1, 10:1 and 5:1) and using a γ counter (PerkinElmer, Waltham, MA).29 To elu- cidate whether PD-L1 blockade can enhance the killing potential of EBV-CTL and EBV-rejT against ENKL, NK-YS cells were cul- tured with 10 mg/mL of anti-PD-L1 antibody (Ultra-LEAFTM puri- fied anti-human CD274, BioLegend) for three days preceding the assay.
Antitumor activity in vivo model
To evaluate the antitumor effects of LMP2-CTL and LMP2-rejT
against ENKL, cells from an HLA class I-matched ENKL line, NK- YS, that had been transduced with a γ-retroviral vector encoding the fusion protein GFP/FFluc were sorted for GFP expression by flow cytometry. Six-week-old female NOD/Shi-scid, IL-2RγKO Jic (NOG) mice (In-Vivo Science, Tokyo, Japan) were engrafted intraperitoneally with NK-YS (1×105 cells/mouse) and tumor growth was monitored using the Xenogen-IVIS Imaging System (Xenogen, Alameda, CA, USA). Once a progressive increase of bioluminescence occurred, usually four days after tumor inocula- tion, mice were treated intraperitoneally with three once-weekly doses of 5×106 LMP2-rejT ± 50 mg of anti-PD-1 Ab or with 5×106 original LMP2-CTL ± 50 mg of anti-PD-1 Ab (In VivoMAb anti-h PD-1, BioXCell, West Lebanon, NH, USA).
PCR and sequencing
EBV strain typing of the NK-YS cells in ascites was performed by PCR using LMP2-specific primers 5’-TATGAATCCAGTAT- GCCTGC-3’ and 5’-CGCAGTAAGCACTGTCACCG-3’ as described29 to detect LMP2 epitopes that are associated with HLA- A*2402.
Results
Distinct PD-L1 expression was significantly associated with poor prognosis in ENKL
To understand the microenvironment of ENKL cells, ENKL cells and tumor-infiltrating lymphocytes (TIL) were analyzed by immunohistochemical staining for PD-L1 and PD-1 receptor expression respectively. As EBV in situ hybridization demonstrated infection in all 28 cases, we initially expected high PD-L1 expression. However, the proportion of ENKL cells (PD-L1 expression ratio, lym- phoma cell : macrophage) was 5-10% (+, positive) in only three samples, 2-3% (+/-, weakly positive) in seven, 1% (- /+, slightly positive) in one, and negative in 17 (Figure 1 A- B).
The presence of many PD-1-expressing TIL is associated with favorable overall survival (OS) in patients with dif- fuse large B-cell lymphoma.26 It is noteworthy that PD-1+ TIL were rarely observed in ENKL, with only 1% of PD-1+ TIL seen in only two of 28 samples (Figure 1 C-D). Table 1 summarizes the characteristics of patients diag- nosed with ENKL in our institute. Three patients whose lymphoma cells expressed PD-L1 (5-10%) were in stage IV
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