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Establishment of a protocol for rapidly expanding Epstein–Barr-virus-specific cytotoxic T cells with enhanced cytotoxicity

BMC Cancer volume 24, Article number: 980 (2024) Cite this article

Abstract

Background

Lytic Epstein–Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs.

Methods

By culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs.

Results

When IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs.

Conclusion

We established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation.

Peer Review reports

Introduction

For patients with unresectable recurrent or metastatic nasopharyngeal carcinoma (R/M NPC), gemcitabine/cisplatin (G/C)-based systemic therapy is recommended as first-line therapy [1]. However, 8.3–9.1% of patients with R/M NPC do not benefit from this regimen [2, 3]. In cases where NPC becomes resistant to G/C, immune checkpoint inhibitor (ICI)-based immunotherapies are an alternative [1]. Yet, only 52.1% of patients with G/C-resistant R/M NPC benefit from ICI-based immunotherapies [4], highlighting an urgent need for more effective therapeutic options for this population.

The adoptive transfer of tumor-specific cytotoxic T cells (CTLs) is a promising approach for treating various cancer types [5]. Given that a high preponderance of NPC cases are associated with Epstein–Barr virus (EBV) infection [6], and considering the successful utilization of EBV-specific CTLs (EBV-CTLs) in treating EBV-associated hematological malignancies [7], utilization of EBV-specific CTLs (EBV-CTLs) in NPC treatment becomes promising.

To generate autologous EBV-CTLs, peripheral blood mononuclear cells (PBMCs) or tumor-infiltrated lymphocytes (TILs) are commonly used as sources due to their existence of EBV-specific effector memory T cells. These cells can be activated by co-culturing with feeder cells such as irradiated EBV-transformed lymphoblastoid cells [EBV-LCLs] or EBV-antigen loaded/expressed dendritic cells [DCs], which facilitates antigen presentation and growth support [8]. However, protocols involving feeder cells for generating EBV-CTLs require 19–31 days, potentially cause substantial delays in NPC treatment [9, 10]. Additionally, gene editing technology, which is involved in generating EBV-expressed DCs, is concerned in clinics by causing genomic instability [11]. These studies underscore the urgent need for developing a safer and more efficient method for producing EBV-CTLs for NPC treatment.

In 2014, Choi et al. and Eom et al. introduced the EBViNT protocol, a two-step protocol for expanding EBV-CTLs. In this protocol, PBMCs are first cultured with EBV-specific antigens for 14 days, after which the EBV-CTLs in these PBMCs are sequentially isolated and expanded [12, 13]. EBViNT uses allogeneic irradiated PBMCs instead of EBV-LCLs during cultivation. However, EBViNT is time-inefficient because EBV-CTL production takes 31 days [13]. The present study introduced an EBV-CTL expansion protocol for rapidly producing EBV-CTLs without feeder cells or T-cell isolation. To develop the protocol, we tested the effectiveness of a combination of interleukin 2 (IL-2), IL-15, and interferon α (IFN-α) in promoting EBV-CTL production; these three cytokines have been documented to promote viral-mediated T-cell response [14, 15]. Additionally, we examined whether our modified EBV-CTL protocol can expand virus-specific CTLs beyond EBV.

Materials and methods

Ethical statement and study design

This study was designed in accordance with the Declaration of Helsinki and received approval from the Institutional Review Board of Far Eastern Memorial Hospital (approval code: 109007-F). PBMCs from healthy donors were used to culture virus-specific CTLs. These donors were required to be older than 20 years old; free from liver, kidney, or hematopoietic abnormalities; and seropositive for serum anti-EBV immunoglobulin G (IgG) or human cytomegalovirus (CMV) IgG. Finally, four healthy subjects participated this study. After a donor signed an informed consent form, we collected 40 mL of peripheral blood for PBMC isolation.

Reagent and antibodies

The reagents and antibodies used in the present study are listed in Supplementary Tables 1 and 2. These reagents and antibodies were aliquoted immediately upon receipt and stored under the recommended conditions until use.

PBMC isolation and CTL cultivation

PBMC isolation was performed using density-gradient centrifugation described in our previous study [16]. Isolated PBMCs were cultured with complete medium (AIM-V medium containing 10% [v/v] of human platelet lysate) with cytokines (human IL-2, IL-15, or IFN-α), and virus-specific peptides (BMLF-1 peptide, EBV peptide pools [EBV-PPs], or CMV peptide pools [CMV-PPs]) supplementation under culture conditions (37 °C, 5% CO2, and saturated humidity). Since Day 3, the cultured medium was supplied or renewed with the complete medium with the indicated cytokine cocktail (without peptide) supplementation every three days to maintain the density of cultured cells at 1 × 106 cells/mL. Cultured cells were scheduled for harvest on Days 6, 9, 12, or 15, and the harvested cells were subjected to functional or phenotypic analysis.

Immunostaining and CTL identification

The protocol of immunostaining is generally available in our previous study [17]. Cultured cells were stimulated with a cell stimulation cocktail for 2 h, after which immunostaining and fluorescent-pattern analysis were conducted using the Navios flow cytometer (Beckman-Coulter, Brea, CA, USA) and Kaluza analysis software (V2.3, Beckman-Coulter). The CTLs in the cultured cells were identified through a pedigree (Supplementary Fig. 1).

Measurement of T-cell expansion and activation

PBMCs were stained with a CellTrace Violet cell proliferation kit (CTV) and cultured under corresponding conditions. Afterward, cells were subjected to immunostaining and fluorescent analysis. The CTV pattern from the cells cultured with medium alone were considered un-proliferated, while cells with a lower CTV intensity were classified as proliferated.

T-cell activation was determined by examining the expression of 4-1BB on T cells, which was assessed through immunostaining [18].

Killing activity assay

We assessed peptide-specific killing activity by co-incubating CTLs with autologous PBMCs loaded with virus-specific peptides (BMLF-1, EBV-PP, or CMV-PP) and measuring apoptotic PBMCs. In brief, autologous PBMCs were incubated with virus-specific peptides and co-incubated with CTLs (effector: target ratio 5:1). Apoptotic analysis was performed using a PanToxiLux kit. The fluorescent pattern from cells PBMCs without CTL co-incubation was categorized as non-apoptotic, and those with higher fluorescence as apoptotic.

Statistical analysis

We independently performed every experiment thrice, and the results are presented in terms of the mean ± standard deviation (SD) and on dot plots (representative once) or bar charts (mean ± SD), which were drawn using Kaluza (dot plot) and Prism (bar chart; V9.0, GraphPad Software, La Jolla, CA, USA) software. Unpaired Student’s t-test and the joint use of one-way analysis of variance (ANOVA) with Dunnett’s test (for comparison with the control group) or Tukey’s test (for multiple comparisons) were conducted to determine statistical significance. P-values less than 0.05, 0.01, 0.001, and 0.0001 are labeled with the symbols *, **, ***, and ****, respectively.

Results

IL-15 synergized with IL-2 to expand CTLs

The primary aim of this study is to develop a GMP-compatible protocol for rapidly expanding high-purity virus-specific CTLs from PBMCs, which can be reached by expanding circulating virus-specific effector memory T cells. Studies have suggested that IL-15 can activate effector memory CTLs and can be utilized in triggering in vitro expanding virus-specific effector memory T cells, though its role in expanding EBV-CTLs was not examined [19]. To determine the potential of IL-15 in promoting CTL expansion, we cultured PBMCs from three unrelated healthy donors carrying anti-EBV IgG in the complete medium with IL-2 or IL-2 plus IL-15 for six days and acquired flow data on Day 6. Literature has suggested that the lytic stage of EBV infection associated with the oncogenesis of EBV-associated solid tumors [20], and HLA-A02:01 (the HLA genotype of the donors)-restricted BMLF-1 antigen had superior activity in inducing EBV-specific T-cell memory than other lytic antigens of EBV [21]. Therefore, we chose BMLF-1 antigen as a target peptide for stimulating EBV-CTL expansion. We compared the percentage of proliferated active CTLs (4-1BB+CD8+) between the IL-2 and IL-2 plus IL-15 groups. The proportion of proliferated active CTLs was 1.2 times higher in the IL-2 plus IL-15 group than in the IL-2 group (Fig. 1A & B). IL-2 plus IL-15 induced a time-dependent increase in the proportion of proliferated active CTLs, which peaked on Day 12 (38.99%) and rapidly decreased thereafter (Fig. 1C, Supplementary Fig. 2). These results suggest that IL-15 can synergize with IL-2 in activating CTLs, which can accelerate the expansion of CTLs.

Fig. 1
figure 1

IL-15 synergizes with IL-2 to expand cytotoxic T lymphocytes (CTLs). The detailed cultured protocol was available in Materials and Methods. Peripheral blood mononuclear cells (PBMCs; density 1 × 106 cells/mL; n = 3) were labeled with CellTrace Violet (CTV) and subjected to culture with complete medium (4% human platelet lysate in AIM-V medium) supplied with IL-2 (40 ng/mL), IL-15 (2.5 ng/mL), and BMLF1 peptide (10 µg/mL) for six days. On Day 6, the cultured cells were harvested and subjected to determine the amount of proliferated activated CTLs (4-1BB+CD8+) using flow cytometry based on the pedigree outlined in Supplementary Fig. 1 (A) for the comparison of the proportion between the IL-2 + IL-15 and IL-2 groups using unpaired Student’s t-tests (B). Afterward, PBMCs were cultured with a complete medium (mentioned above) supplied with cytokines and BMLF-1 peptide for 15 days, and the monitor of activated CTLs was scheduled on the indicated days (C). ****, p < 0.0001

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IFN-α synergized with T-cell receptor engagement to enhance expansion and virus-specific cytotoxicity of cultured CTLs

The literature suggests that type I interferon (IFN-α/β) can support T-cell activation after T-cell receptor (TCR) engagement [22]. Therefore, we assessed the potential of IFN-α in enhancing CTL expansion through culturing PBMCs from three unrelated healthy donors with IL-2, IL-15, BMLF-1 peptide, and IFN-α and sought the proper treating duration of IFN-α if which can enhance the CTL expansion. Given that the peak proportion of activated EBV-CTL proportion was on Day 9 when IFN-α was applied in the EBV-CTL expansion (Supplementary Fig. 3), we shifted the cultivation period from 12 days to 9 days and acquired the data on Day 9. In consideration of the in-sequence interaction of the signaling between IFN-α and TCR engagement [23], we applied IFN-α on two specific days (days 0 and 3) for a duration of 3, 6, or 9 days based on IL-2/IL-15-mediated CTL expansion and acquired data on Day 9 (Fig. 2A). Implementing IFN-α treatment from day 0 resulted in the IFN-α treatment group yielding 1.5 times more proliferated active CTLs relative to the non-IFN-α treatment (p < 0.01; Fig. 2B). The amount of proliferated active CTLs after implementing IFN-α from day 3 was similar to that in the non-IFN-α treatment group (p > 0.05; Fig. 2B). This finding suggests that IFN-α treatment should be concurrent with TCR engagement. Of note, the proportion of proliferated active CTLs did not show a time-dependent increase with the duration of IFN-α treatment (p > 0.05), suggesting that period of IFN-α treatment is not a crucial factor for promoting EBV-CTL expansion. Nevertheless, the proportion of proliferated active CTLs obtained after 6 days of IFN-α treatment exhibited less variation among the repeated experiments compared with the other two groups (Fig. 2B), suggesting that 6 days of IFN-α treatment caused more consistent production of active CTLs than 3 or 9 days of IFN-α treatment.

Fig. 2
figure 2

Interferon-α (IFN-α) treatment concurrent with TCR engagement promotes CTL activation. The schedule of IFN-α supplementation during the CTL cultivation is represented in (A). Briefly, PBMCs (n = 3, density 1 × 106 cells/mL) were labeled with CTV and subjected to EBV-CTL expansion via the protocol in Materials and Methods for nine days. During the cultivation, IFN-α supplementation was scheduled in terms of experimental designation. On Day 9, the cultured cells were harvested and subjected to analyze phenotypic data by flow cytometry based on the pedigree outlined in Supplementary Fig. 1 (B). Results were shown in mean ± SD with a bar chart. One-way ANOVA with Dunnett’s test for post hoc analysis was for calculating the statistical significance of each comparison. **, p < 0.01; ***, p < 0.001; ns, not significant

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We further investigated the antigen specificity of the activated CTLs described in the previous paragraph. By labeling CTLs with allophycocyanin-labeled BMLF-1 peptide pentamer, the proportions of BMLF-1-reactive CTLs with BMLF-1-free, BMLF-1, and IFN-α-treated BMLF-1 peptide were determined to be 1.97%, 18.01%, and 50.69% (16.1% of total amount of CTLs), respectively (p < 0.01, Fig. 3A & B). This finding suggests that the implementation of IFN-α in CTL expansion can enhance the expansion of virus-specific CTLs. To evaluate the cytokine secretory change of IFN-α treatment during CTL-expansion, we cultured PBMCs with BMLF-1, IFN-α, or BMLF-1/IFN-α for 9 days followed by measuring the expression of secretory cytokines (IFN-γ and perforin) and de-granulating molecules (CD107a) in the activated CTLs. CTLs without BMLF-1 treatment presented comparable expression levels of secretory cytokines and de-granulating molecules to those without BMLF-1 and IFN-α treatment (Fig. 3B & C). CTLs treated with BMLF-1 exhibited increased expression of secretory cytokines and de-granulating molecules. Combining BMLF-1 and IFN-α, the expression of secretory cytokines and de-granulating molecules in EBV-CTLs were further augmented (Fig. 3A). These results indicated that utilizing IFN-α in CTL cultivation can potentiate the cytokine-secretory activity of CTLs. The CTLs from the BMLF-1 peptide plus IFN-α group induced apoptosis in 29.45% of the BMLF-1-labeled autologous PBMCs, which was significantly higher than that achieved by the BMLF-1 peptide group (p < 0.01; Fig. 3D & E). This finding suggests a higher level of BMLF-1-specific cytotoxicity in the CTLs obtained through BMLF-1 peptide plus IFN-α treatment than in those obtained through BMLF-1 peptide treatment. In summary, the implementation of IFN-α concurrent with TCR engagement during CTL cultivation promotes virus-specific CTL expansion and strengthens the antigen-specific cytotoxicity of cultured CTLs. Culturing PBMCs with IL-2, IL-15, and IFN-α can yield high-purity EBV-CTLs in 9 days. We named this EBV-CTL-expanding protocol “EBaT8.” The applicability of EBaT8 in expanding other virus-specific CTLs is discussed in the following section.

Fig. 3
figure 3

Using IFN-α in CTL expansion enhances EBV-specificity of cultured CTLs. EBV-CTLs were expanded from PBMCs (n = 4, density 1 × 106 cells/mL) by the protocol in Materials and Methods for nine days. IL-2 and IL-15 were supplied across the cultivation, and IFN-α was between Day 0 and Day 6. On Day 9, the cultured cells were harvested and subjected to acquire the data regarding the amount of BMLF-1-reactive activated CTLs (A & B), expression of secretory cytokines (perforin and IFN-γ), degranulating molecule (CD107a) (C), and BMLF-1-specific cytotoxicity (D & E), via flow cytometry. Results were presented in a representative once and a bar chart with the mean ± SD from three independent experiments. One-way ANOVA with Tukey’s test for post hoc analysis was for calculating the statistical significance of each comparison. *, p < 0.05; **, p < 0.01; ***, p < 0.001

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Using EBaT8 to expand EBV- and CMV-specific CTLs obtained from PBMCs

To determine the feasibility of the EBaT8 protocol for expanding EBV-specific (not only BMLF-1) and CMV-specific CTLs, we applied the EBaT8 with EBV-PPs (comprising 43 MHC-I/II-restricted peptides) to PBMCs from three unrelated healthy donors followed by examining the EBV-specific cytotoxicity of cultured CTLs. The number of activated CTLs was higher in the EBV-PP treatment group than in the non-EBV-PP treatment group (Fig. 4A). Co-incubating EBV-PP-labeled PBMCs with autologous CTLs subjected to EBV-PP treatment resulted in a significantly higher number of apoptotic PBMCs relative to co-incubation with autologous CTLs without EBV-PP treatment (Fig. 4B & C). This finding indicates that the EBaT8 protocol can be applied to expand EBV-specific CTLs other than BMLF-1.

Fig. 4
figure 4

EBV pooled peptides can be used in EBaT8 protocol to expand EBV-specific CTLs. PBMCs (n = 3) were cultured with the protocol described in Fig. 3 for 9 days and used EBV peptide pools (EBV-PPs) instead of BMLF-1 peptide during cultivation. On Day 9, cultured cells were harvested and proceeded to evaluate the proportion of total/activated CTLs (A) and measure the EBV-specific killing activity against EBV-PP-loaded autologous PBMCs (B & C). Data of CTL proportion and killing activity were acquired by flow cytometry on Day 9. Results are presented in both representative manner and bar chart with the mean ± SD from three independent experiments. One-way ANOVA with Tukey’s test for post hoc analysis was for calculating the statistical significance of each comparison. **, p < 0.01

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Then, we modified the EBaT8 protocol by replacing EBV-PPs with CMV-PPs (PepTivator CMV pp65) and using them in CMV-specific CTL cultivation from three unrelated healthy donors to evaluate the applicability of EBaT8 in expanding virus-specific CTLs other than EBV. In the preliminary test, the proportion of CMV-specific CTLs peaked on Day 6 rather than Day 9 (data not shown). Therefore, we shortened the culturing period of the EBaT8 protocol from 9 to 6 days for CMV-specific CTLs. The amount of proliferated activated CTLs obtained from the CMV-PP treatment group was higher than that obtained from the non-CMV-PP treatment group (Fig. 5A). Furthermore, the CTLs obtained from the CMV-PP treatment group exhibited higher CMV-specific cytotoxicity against CMV-PP-labeled PBMCs than those obtained from the non-CMV-PP treatment group (Fig. 5B & C). This finding suggests that the EBaT8 protocol can expand virus-specific CTLs ex vivo, not only EBV-specific but also CMV-specific.

Fig. 5
figure 5

CMV-specific peptide pools can be used in EBaT8 protocol to expand CMV-specific CTLs. PBMCs (n = 3) were cultured with the protocol described in Fig. 3 for 6 days and used CMV peptide pools (CMV-PPs) instead of BMLF-1 peptide during cultivation. On Day 9, cultured cells were harvested and proceeded to evaluate the proportion of total/activated CTLs (A) and measure the CMV-specific killing activity against CMV-PP-loaded autologous PBMCs (B & C). Data of CTL proportion and killing activity were acquired by flow cytometry on Day 6. Results are presented in both representative manner and bar chart with the mean ± SD from three independent experiments. One-way ANOVA with Tukey’s test for post hoc analysis was for calculating the statistical significance of each comparison. **, p < 0.01

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Discussion

In this study, we developed a novel protocol (EBaT8) for the ex vivo expanding EBV-specific CTLs from PBMCs in 9 days. In the EBaT8 protocol, IL-15 is synergized with IL-2 to induce antigen-mediated CTL growth. Concurrent IFN-α treatment and TCR engagement further enhance EBV-specific CTL growth and their EBV-specific cytotoxicity. Additionally, the successful expansion of the CMV-specific CTLs by the EBaT8 protocol suggested that the EBaT8 can extend to expand other virus-specific CTLs.

We established a one-step EBV-CTL-expanding protocol from PBMCs using EBV-specific antigens and several cytokines to promote T-cell activation. Given around 50% objective response rate in EBV-CTL treatment against EBV-associated hematological malignancies [24, 25], the European Medical Agency has also approved the introduction of Tabelecleucel, the first EBV-CTL product for the allogenic treatment of these malignancies, to the market in 2022 [26]. Typically, autologous EBV-CTLs are generated using EBV-LCLs or DCs from PBMCs as antigen-presenting cells for activating effector memory T cells [27]. However, generating autologous EBV-LCLs requires 3 to 4 weeks, potentially delaying the treatment plan due to the rapid progression of EBV-associated malignancies. The DC-mediated EBV-CTL production takes 7 to 25 days, which in some cases is shorter than the EBV-LCL-based protocol, but requires cost-ineffective purification of DCs and T cells [27]. Thus, a method for rapidly producing autologous EBV-CTLs is urgently needed. On the basis of EBV-LCL-based culturing protocols, Choi et al. and Eom et al. reported a modified EBV-CTL-production protocol called EBViNT. In EBViNT, autologous PBMCs are cultured with EBV-specific antigens and IL-2 for 14 days, then activated CTLs (4-1BB+CD8+) were immobilized on plate coated with anti-4-1-BB antibodies and cultured with irradiated allogeneic PBMCs for another 14 days [12, 13]. Despite the EBViNT protocol using endogenous DCs from PBMCs for antigen presentation instead of EBV-LCLs, the EBViNT still requires a production period of 31 days [12]. Moreover, the EBV-CTLs and activated CTLs among the EBViNT-produced CTLs exhibit purity levels of approximately 11.9% and 2.6%, respectively, which can be enhanced [13]. Gary et al. demonstrated that EBV-CTLs can be produced by directly culturing PBMCs with EBV-specific antigens and IL-2 in 9 days, archiving 43.3% purity of EBV-CTLs in activated CTLs (CD62L+) [28]. This result suggests that several culture components or methods, such as purification of activated CTLs, EBV-LCLs, or irradiated allogeneic PBMCs, are unnecessary. We enhanced the protocol proposed by Gary et al. by adding IFN-α and IL-15 to potentiate productivity and virus-specific cytotoxicity. The purity of EBV-CTLs in total activated CTLs from EBaT8 was 50.69% (Fig. 3A & B), higher than the protocol from Gary et al.

We confirmed that IL-15 can synergize with IL-2 to stimulate EBV-CTL proliferation. Baltaleucel T, an investigational autologous EBV-CTL product by Cell Medica, uses IL-15 to accelerate monocyte maturation into DCs and promote their immunomodulatory effect [25, 29, 30]. Hansen et al. showed that IL-15 supports central memory T-cell expansion and can be utilized to expand CMV-CTLs [31]. IL-15 treatment reduces the TCR threshold in memory CTLs by triggering a gene expression profile similar to TCR engagement [32, 33]. In addition, IL-15 promotes IL-2 production and enhances its proliferative efficacy in effector memory CTLs [34]. These results suggest that IL-15 benefits the expansion of CTLs, confirmed in the present study. Additionally, various cytokines synergize with IL-2 (e.g., IL-7, IL-12, and IL-21) in promoting CTL proliferation or cytotoxicity, and such synergize can potentially potentiate EBV-CTL production [35,36,37]. Of note, binding IL-15 with IL-15 receptor α (IL-15Rα, either membrane-bound or soluble form) strengthens the tumor-control activity [38], suggesting that using IL-15/IL-15Rα complex or fusion protein (e.g., IL-15/IL-15RA-Fc) may be superior to IL-15 alone [39].

The present study highlights the benefits of using IFN-α in EBV-CTL production, marking the first application of type 1 interferons (IFN-Is) to boost EBV-CTL production. IFN-Is (comprising IFN-α, β, ε, κ, and ω) trigger monocytes differentiating into DCs and promote DC antigen-presenting activity in cross-presentation [40, 41]. Additionally, IFN-Is promote DCs releasing IL-15/IL-15Rα complex and elevate TCR signaling, thereby increasing the cytotoxicity of CTLs by upregulating granzyme B expression [23, 42, 43]. Sikora et al. found that C57BL/6 mice treated with IFN-α and gp10025 − 33 peptide had higher quantities and antitumor activity of gp100-specific CTLs than those without IFN-α treatment [44]. These studies suggest that IFN-Is can feasibly expand CTLs. We identified that IFN-I treatment since Day 0 yielded a higher proportion of EBV-CTLs than without IFN-I treatment and since Day 3 (Fig. 2). Prolonged IFN-I treatment did not significantly increase CTL yield (Fig. 2B), suggesting that the IFN-I treatment mainly contributes to the antigen presentation of DCs rather than CTL activation. Of note, Sumida et al. reported that IFN-I-treated T lymphocytes expressed lower levels of co-inhibitory molecules (PD-1, Tim-3, and Lag-3) due to down-regulation of TIGIT (i.e., T-cell immunoreceptor with Ig and ITIM domains) [45], suggesting that CTLs produced with IFN-I-based protocol are potentially less sensitive to inhibitory tumor microenvironment, despite prolonged IFN-I treatment not increasing the EBV-CTLs yield.

In addition to the cytokines tested in the present study, IL-4 and IL-7 are considered to boost the yield of cultured CTL. IL-7, in cooperation with IL-15, maintains memory-T-cell survival after contraction and increases their number and IFN-γ secretory activity [46,47,48,49]. Additionally, IL-7 can activate plasmacytoid dendritic cells, enhancing memory-CTL-activating activity by increasing IFN-I secretion [50]. These studies imply that IL-7 may effectively promote the CTL yield in vitro expansion. IL-4, like IL-7, maintains memory CTL survival by downregulating pro-apoptotic signaling [51]. However, IL-4 generally promotes virtual-memory-CTL proliferation rather than true-memory CTLs [52, 53]. Additionally, IL-4/7 combination predominantly promotes CD4 T cell proliferation instead of CD8 T cells even though antigen-specific promotion of IL-4/7 is comparable with IL-15 alone or IL-6/15 combination [54], suggesting that IL-4 and IL-7 may not be suitable for in vitro expanding CTLs without further purification.

The present study introduced the EBaT8 protocol, a streamlined one-step protocol for rapidly producing EBV-CTLs from PBMCs without feeder cells or purification. This novel protocol is also applicable in expanding virus-specific CTLs beyond EBV-CTLs. In future studies, we aim to assess the therapeutic activity of EBV-CTLs from EBaT8 against EBV-associated malignancies, such as nasopharyngeal carcinoma and post-transplanted lymphoproliferative disorder, in a clinical setting.

Data availability

The data generated in the present study may be requested from the corresponding author.

References

  1. Bossi P, Chan AT, Even C, Machiels JP. clinicalguidelines@esmo.org EGCEa: ESMO-EURACAN Clinical Practice Guideline update for nasopharyngeal carcinoma: adjuvant therapy and first-line treatment of recurrent/metastatic disease. Ann Oncol. 2023;34(3):247–50.

    Article  CAS  PubMed  Google Scholar 

  2. Hsieh JC, Hsu CL, Ng SH, Wang CH, Lee KD, Lu CH, Chang YF, Hsieh RK, Yeh KH, Hsiao CH, et al. Gemcitabine plus cisplatin for patients with recurrent or metastatic nasopharyngeal carcinoma in Taiwan: a multicenter prospective phase II trial. Jpn J Clin Oncol. 2015;45(9):819–27.

    Article  PubMed  Google Scholar 

  3. Yang Q, Nie YH, Cai MB, Li ZM, Zhu HB, Tan YR. Gemcitabine Combined with Cisplatin has a better effect in the treatment of Recurrent/Metastatic Advanced Nasopharyngeal Carcinoma. Drug Des Devel Ther. 2022;16:1191–8.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Xu JY, Wei XL, Wang YQ, Wang FH. Current status and advances of immunotherapy in nasopharyngeal carcinoma. Ther Adv Med Oncol. 2022;14:17588359221096214.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Liu Q, Li J, Zheng H, Yang S, Hua Y, Huang N, Kleeff J, Liao Q, Wu W. Adoptive cellular immunotherapy for solid neoplasms beyond CAR-T. Mol Cancer. 2023;22(1):28.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Khan G, Fitzmaurice C, Naghavi M, Ahmed LA. Global and regional incidence, mortality and disability-adjusted life-years for Epstein-Barr virus-attributable malignancies, 1990–2017. BMJ Open. 2020;10(8):e037505.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Heslop HE, Sharma S, Rooney CM. Adoptive T-Cell therapy for Epstein-Barr Virus-related Lymphomas. J Clin Oncol. 2021;39(5):514–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. He J, Tang XF, Chen QY, Mai HQ, Huang ZF, Li J, Zeng YX. Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy. Chin J Cancer. 2012;31(6):287–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pender MP, Csurhes PA, Smith C, Douglas NL, Neller MA, Matthews KK, Beagley L, Rehan S, Crooks P, Hopkins TJ et al. Epstein-Barr virus-specific T cell therapy for progressive multiple sclerosis. JCI Insight 2018, 3(22).

  10. Cooper RS, Kowalczuk A, Wilkie G, Vickers MA, Turner ML, Campbell JDM, Fraser AR. Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy. Clin Exp Immunol. 2021;206(1):68–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Papathanasiou S, Markoulaki S, Blaine LJ, Leibowitz ML, Zhang CZ, Jaenisch R, Pellman D. Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing. Nat Commun. 2021;12(1):5855.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Eom HS, Choi BK, Lee Y, Lee H, Yun T, Kim YH, Lee JJ, Kwon BS. Phase I clinical trial of 4-1BB-based adoptive T-Cell therapy for Epstein-Barr Virus (EBV)-positive tumors. J Immunother. 2016;39(3):140–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Choi BK, Lee SC, Lee MJ, Kim YH, Kim YW, Ryu KW, Lee JH, Shin SM, Lee SH, Suzuki S, et al. 4-1BB-based isolation and expansion of CD8 + T cells specific for self-tumor and non-self-tumor antigens for adoptive T-cell therapy. J Immunother. 2014;37(4):225–36.

    Article  CAS  PubMed  Google Scholar 

  14. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J Exp Med. 2005;202(5):637–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Verbist KC, Klonowski KD. Functions of IL-15 in anti-viral immunity: multiplicity and variety. Cytokine. 2012;59(3):467–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee JM, Chen MH, Chou KY, Chao Y, Chen MH, Tsai CY. Novel immunoprofiling method for diagnosing SLE and evaluating therapeutic response. Lupus Sci Med 2022, 9(1).

  17. Lee JM, Hung YP, Chou KY, Lee CY, Lin SR, Tsai YH, Lai WY, Shao YY, Hsu C, Hsu CH, et al. Artificial intelligence-based immunoprofiling serves as a potentially predictive biomarker of nivolumab treatment for advanced hepatocellular carcinoma. Front Med (Lausanne). 2022;9:1008855.

    Article  PubMed  Google Scholar 

  18. Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood. 2018;131(1):49–57.

    Article  CAS  PubMed  Google Scholar 

  19. Pilipow K, Roberto A, Roederer M, Waldmann TA, Mavilio D, Lugli E. IL15 and T-cell stemness in T-cell-based Cancer Immunotherapy. Cancer Res. 2015;75(24):5187–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Xu X, Zhu N, Zheng J, Peng Y, Zeng MS, Deng K, Duan C, Yuan Y. EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation. PLoS Pathog. 2024;20(1):e1011934.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Catalina MD, Sullivan JL, Bak KR, Luzuriaga K. Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection. J Immunol. 2001;167(8):4450–7.

    Article  CAS  PubMed  Google Scholar 

  22. Huber JP, Farrar JD. Regulation of effector and memory T-cell functions by type I interferon. Immunology. 2011;132(4):466–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Crouse J, Kalinke U, Oxenius A. Regulation of antiviral T cell responses by type I interferons. Nat Rev Immunol. 2015;15(4):231–42.

    Article  CAS  PubMed  Google Scholar 

  24. Prockop S, Mahadeo KM, Beitinjaneh A, Choquet S, Stiff P, Reshef R, Satyanarayana G, Dahiya S, Parmar H, Ye W, et al. Multicenter, Open-Label, phase 3 study of Tabelecleucel for solid organ or allogeneic hematopoietic cell transplant recipients with Epstein-Barr Virus-Driven Post Transplant Lymphoproliferative Disease after failure of Rituximab or Rituximab and Chemotherapy (ALLELE). Blood. 2021;138(Supplement 1):301–301.

    Article  Google Scholar 

  25. Kim WS, Oki Y, Kim SJ, Yoon SE, Ardeshna KM, Lin Y, Ruan J, Porcu P, Brammer JE, Jacobsen ED, et al. Autologous EBV-specific T cell treatment results in sustained responses in patients with advanced extranodal NK/T lymphoma: results of a multicenter study. Ann Hematol. 2021;100(10):2529–39.

    Article  CAS  PubMed  Google Scholar 

  26. Keam SJ. Tabelecleucel: first approval. Mol Diagn Ther. 2023;27(3):425–31.

    Article  CAS  PubMed  Google Scholar 

  27. Zhang Y, Lyu H, Guo R, Cao X, Feng J, Jin X, Lu W, Zhao M. Epstein–Barr virus-associated cellular immunotherapy. Cytotherapy. 2023;25(9):903–12.

    Article  CAS  PubMed  Google Scholar 

  28. Gary R, Aigner M, Moi S, Schaffer S, Gottmann A, Maas S, Zimmermann R, Zingsem J, Strobel J, Mackensen A, et al. Clinical-grade generation of peptide-stimulated CMV/EBV-specific T cells from G-CSF mobilized stem cell grafts. J Transl Med. 2018;16(1):124.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Anguille S, Smits EL, Cools N, Goossens H, Berneman ZN, Van Tendeloo VF. Short-term cultured, interleukin-15 differentiated dendritic cells have potent immunostimulatory properties. J Transl Med. 2009;7:109.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Saikh KU, Kissner TL, Nystrom S, Ruthel G, Ulrich RG. Interleukin-15 increases vaccine efficacy through a mechanism linked to dendritic cell maturation and enhanced antibody titers. Clin Vaccine Immunol. 2008;15(1):131–7.

    Article  CAS  PubMed  Google Scholar 

  31. Hasan AN, Selvakumar A, Shabrova E, Liu XR, Afridi F, Heller G, Riviere I, Sadelain M, Dupont B, O’Reilly RJ. Soluble and membrane-bound interleukin (IL)-15 Ralpha/IL-15 complexes mediate proliferation of high-avidity central memory CD8(+) T cells for adoptive immunotherapy of cancer and infections. Clin Exp Immunol. 2016;186(2):249–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Deshpande P, Cavanagh MM, Le Saux S, Singh K, Weyand CM, Goronzy JJ. IL-7- and IL-15-mediated TCR sensitization enables T cell responses to self-antigens. J Immunol. 2013;190(4):1416–23.

    Article  CAS  PubMed  Google Scholar 

  33. Liu K, Catalfamo M, Li Y, Henkart PA, Weng NP. IL-15 mimics T cell receptor crosslinking in the induction of cellular proliferation, gene expression, and cytotoxicity in CD8 + memory T cells. Proc Natl Acad Sci U S A. 2002;99(9):6192–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mathieu C, Beltra JC, Charpentier T, Bourbonnais S, Di Santo JP, Lamarre A, Decaluwe H. IL-2 and IL-15 regulate CD8 + memory T-cell differentiation but are dispensable for protective recall responses. Eur J Immunol. 2015;45(12):3324–38.

    Article  CAS  PubMed  Google Scholar 

  35. Liu Y, Adu-Berchie K, Brockman JM, Pezone M, Zhang DKY, Zhou J, Pyrdol JW, Wang H, Wucherpfennig KW, Mooney DJ. Cytokine conjugation to enhance T cell therapy. Proc Natl Acad Sci U S A. 2023;120(1):e2213222120.

    Article  CAS  PubMed  Google Scholar 

  36. Battaglia A, Buzzonetti A, Baranello C, Fanelli M, Fossati M, Catzola V, Scambia G, Fattorossi A. Interleukin-21 (IL-21) synergizes with IL-2 to enhance T-cell receptor-induced human T-cell proliferation and counteracts IL-2/transforming growth factor-beta-induced regulatory T-cell development. Immunology. 2013;139(1):109–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sin JI, Kim J, Pachuk C, Weiner DB. Interleukin 7 can enhance antigen-specific cytotoxic-T-lymphocyte and/or Th2-type immune responses in vivo. Clin Diagn Lab Immunol. 2000;7(5):751–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hong E, Usiskin IM, Bergamaschi C, Hanlon DJ, Edelson RL, Justesen S, Pavlakis GN, Flavell RA, Fahmy TM. Configuration-dependent presentation of multivalent IL-15:IL-15Ralpha enhances the Antigen-specific T cell response and anti-tumor immunity. J Biol Chem. 2016;291(17):8931–50.

    Article  CAS  PubMed  Google Scholar 

  39. Xu H, Buhtoiarov IN, Guo H, Cheung NV. A novel multimeric IL15/IL15Ralpha-Fc complex to enhance cancer immunotherapy. Oncoimmunology. 2021;10(1):1893500.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Collin M, Bigley V. Human dendritic cell subsets: an update. Immunology. 2018;154(1):3–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Schiavoni G, Mattei F, Gabriele L. Type I interferons as stimulators of DC-Mediated cross-priming: impact on Anti-tumor Response. Front Immunol. 2013;4:483.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Lu C, Klement JD, Ibrahim ML, Xiao W, Redd PS, Nayak-Kapoor A, Zhou G, Liu K. Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes. J Immunother Cancer. 2019;7(1):157.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Anthony SM, Howard ME, Hailemichael Y, Overwijk WW, Schluns KS. Soluble interleukin-15 complexes are generated in vivo by type I interferon dependent and independent pathways. PLoS ONE. 2015;10(3):e0120274.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Sikora AG, Jaffarzad N, Hailemichael Y, Gelbard A, Stonier SW, Schluns KS, Frasca L, Lou Y, Liu C, Andersson HA, et al. IFN-alpha enhances peptide vaccine-induced CD8 + T cell numbers, effector function, and antitumor activity. J Immunol. 2009;182(12):7398–407.

    Article  CAS  PubMed  Google Scholar 

  45. Sumida TS, Dulberg S, Schupp JC, Lincoln MR, Stillwell HA, Axisa PP, Comi M, Unterman A, Kaminski N, Madi A, et al. Type I interferon transcriptional network regulates expression of coinhibitory receptors in human T cells. Nat Immunol. 2022;23(4):632–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hashimoto M, Im SJ, Araki K, Ahmed R. Cytokine-mediated regulation of CD8 T-Cell responses during Acute and chronic viral infection. Cold Spring Harb Perspect Biol 2019, 11(1).

  47. Xia CS, Long Y, Liu Y, Alifu A, Zeng X, Liu C. IL-7 promotes the expansion of circulating CD28- cytotoxic T lymphocytes in patients with IgG4-Related Disease via the JAK signaling. Front Immunol. 2022;13:922307.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Nanjappa SG, Walent JH, Morre M, Suresh M. Effects of IL-7 on memory CD8 T cell homeostasis are influenced by the timing of therapy in mice. J Clin Invest. 2008;118(3):1027–39.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Huang Y, Zheng H, Zhu Y, Hong Y, Zha J, Lin Z, Li Z, Wang C, Fang Z, Yu X, et al. Loss of CD28 expression associates with severe T-cell exhaustion in acute myeloid leukemia. Front Immunol. 2023;14:1139517.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Pandit H, Valentin A, Angel M, Deleage C, Bergamaschi C, Bear J, Sowder R, Felber BK, Pavlakis GN. Step-dose IL-7 treatment promotes systemic expansion of T cells and alters immune cell landscape in blood and lymph nodes. iScience. 2023;26(2):105929.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Silva-Filho JL, Caruso-Neves C, Pinheiro AAS. IL-4: an important cytokine in determining the fate of T cells. Biophys Rev. 2014;6(1):111–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Park HJ, Choi EA, Choi SM, Choi YK, Lee JI, Jung KC. IL-4/IL-4 ab complex enhances the accumulation of both antigen-specific and bystander CD8 T cells in mouse lungs infected with influenza a virus. Lab Anim Res. 2023;39(1):32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Renkema KR, Lee JY, Lee YJ, Hamilton SE, Hogquist KA, Jameson SC. IL-4 sensitivity shapes the peripheral CD8 + T cell pool and response to infection. J Exp Med. 2016;213(7):1319–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Lazarski CA, Datar AA, Reynolds EK, Keller MD, Bollard CM, Hanley PJ. Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay. Cytotherapy. 2021;23(1):65–76.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This manuscript was edited by Wallace Academic Editing.

Funding

This study is fully sponsored by FullHope Biomedical Co., Ltd.

Author information

Authors and Affiliations

  1. FullHope Biomedical Co., Ltd., 10F., No. 10, Ln. 609, Sec. 5, Chongxin Rd., Sanchong Dist., New Taipei City, 241405, Taiwan

    Chih-Hao Fang, Ya Fang Cheng, Shian-Ren Lin, Wan-Yu Lai & Jan-Mou Lee

  2. Department of Otolaryngology, Far Eastern Memorial Hospital, New Taipei City, 220216, Taiwan

    Li-Ren Liao

  3. Division of Nephrology, Department of Medicine, Far Eastern Memorial Hospital, New Taipei City, 220216, Taiwan

    Yen-Ling Chiu

  4. Department of Medical Research, Far Eastern Memorial Hospital, No. 121, Sec. 2, Nanya S. Rd., Banqiao Dist., New Taipei City, 220216, Taiwan

    Yen-Ling Chiu

  5. Graduate Institute of Medicine and Graduate Program in Biomedical Informatics, Yuan Ze University, Taoyuan, 320315, Taiwan

    Yen-Ling Chiu

  6. Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, 100233, Taiwan

    Yen-Ling Chiu

Authors
  1. Chih-Hao Fang
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  4. Wan-Yu Lai
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  7. Jan-Mou Lee
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Contributions

Jan-Mou Lee and Yen-Ling Chiu conceived and supervised the study. Chih-Hao Fang and Ya Fang Cheng conducted the investigation and data plotting. Wan-Yu Lai and Li-Ren Liao managed donor enrollment. Shian-Ren Lin wrote the original draft. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to Yen-Ling Chiu or Jan-Mou Lee.

Ethics declarations

Ethics approval and consent to participate

The designation of this study complied with the Declaration of Helsinki. The study protocol was reviewed and approved by the Institutional Review Board of Far Eastern Memorial Hospital (approval code: 109007-F). All participants signed the informed consent form before blood collection.

Patient consent for publication

Not applicable.

Competing interests

EBaT8 is patent filed by FullHope Biomedical Co., Ltd. in Taiwan (patent no I840994). Chih-Hao Fang, Ya Fang Cheng, Shian-Ren Lin, and Wan-Yu Lai are employees of FullHope Biomedical Co., Ltd. Jan-Mou Lee is the CEO of FullHope Biomedical. Co., Ltd. In addition to the above, the authors declare no other conflict of interest to this study.

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Fang, CH., Cheng, Y.F., Lin, SR. et al. Establishment of a protocol for rapidly expanding Epstein–Barr-virus-specific cytotoxic T cells with enhanced cytotoxicity. BMC Cancer 24, 980 (2024). https://doi.org/10.1186/s12885-024-12707-7

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Keywords

BMC Cancer

ISSN: 1471-2407

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