Spontaneous EBV-positive B cell line from rectal tumor: characterization, interaction with autologous tumor-infiltrating T lymphocytes, comparison with B lymphoma cell lines

Spontaneous EBV-positive B cell line from rectal tumor: characterization, interaction with autologous tumor-infiltrating T lymphocytes, comparison with B lymphoma cell lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Spontaneous EBV-positive B cell line from rectal tumor: characterization, interaction with autologous tumor-infiltrating T lymphocytes, comparison with B lymphoma cell lines Tatiana V. Petrova, Daria V. Kuznetzova, Alexandra V. Kanygina, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6835907/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Recent studies underscore herpesvirus-associated risks, especially HHV6 and Epstein–Barr virus (EBV), in CAR-T and tumor-infiltrating lymphocyte (TIL) therapies, partly due to ex vivo cell manipulation. EBV-driven B cell outgrowth can occur during TIL expansion, and EBV-transformed B cells may exert immunomodulatory functions, potentially impacting TIL therapy efficacy — areas that remain underexplored. Here, we characterized a spontaneously arising wild-type EBV-transformed B cell line, lcl_burn0214, derived from rectal cancer tumor tissue during ex vivo culture. The lcl_burn0214 cell line has undergone 70 passages and was confirmed as a monoclonal B cell line of patient origin via HLA genotyping, immunophenotyping (CD19, CD20), B cell receptor clonotype analysis. RNA-seq analysis comparing EBV-positive and -negative B lymphoma and lymphoblastoid cell lines revealed that lcl_burn0214’s gene expression closely resembles that of lymphoblastoid cells rather than malignant B lymphomas. In immunocompromised mice, lcl_burn0214 exhibited limited tumorigenicity. The cell line expressed immune checkpoint genes (CD274, CSF1, TNFSF4). Nevertheless, autologous TILs were negative for B cells and exhibited specific anti-EBV activity, as demonstrated by the interferon-gamma response in the ELISPOT assay during coculture with lcl_burn0214. Our findings underscore the importance of monitoring TIL products for EBV-positive B cell contamination during manufacturing to ensure safety and therapeutic efficacy. Biological sciences/Immunology/Immunotherapy Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Rectal cancer Biological sciences/Microbiology/Virology/Herpes virus Figures Figure 1 Figure 2 Figure 3 Introduction EBV, a member of the herpesvirus family, primarily infects B lymphocytes and persists as an episome throughout the host's lifespan, evading T cell-mediated immune responses. EBV is associated with B cell lymphomas and can trigger post-transplant lymphoproliferative disease in immunocompromised individuals [1]. Recent studies have raised concerns about herpesvirus-associated risks in T cell therapies, including chimeric antigen receptor (CAR) T cell and tumor-infiltrating T lymphocyte treatments. Latent human herpesvirus 6 reactivation has been demonstrated in CAR-T cells in vivo, along with a reported case of EBV-positive B lymphoma following TIL therapy for metastatic melanoma [2, 3]. Previous studies have reported the spontaneous ex vivo outgrowth of immortalized EBV-transformed B cell lines from various solid and hematological tumors, including ovarian cancer, colorectal cancer, and leukemia [4–6]. Several animal studies have demonstrated that highly immunocompromised mice engrafted with patient tumor tissues—including ovarian, gastric, breast, lung, and colorectal tumors—often develop metastatic EBV-positive B cell lymphocytic tumors rather than patient-derived epithelial tumor xenografts [7–10]. Tumor fragments used to generate cell products for tumor-infiltrating lymphocyte (TIL) therapy contain diverse cell populations, such as TILs and tumor-infiltrating B lymphocytes (B-TILs) [11]. Therefore, during ex vivo TIL expansion for cell therapy, there is a risk that EBV-transformed B-TILs may proliferate concurrently, particularly in colorectal cancer, where EBV-positive lymphocyte infiltration of the tumor occurs in approximately 40% of cases [12]. Also EBV-transformed B cells have the potential to modulate immune responses by reshaping T cell receptor repertoire [10]. These features of EBV-transformed B-TILs may significantly influence the efficacy and safety of TIL therapies, but remain insufficiently explored. In this study, we characterized a wild-type EBV-transformed B cell line, lcl_burn0214, derived spontaneously from a rectal cancer specimen during ex vivo culture. Using RNA-seq, we compared its gene expression with established EBV-positive and EBV-negative B lymphoma lines and B95-8 EBV-infected lymphoblastoid cell lines (LCLs, B95-8_LCLs). We then assessed possible immune regulating features of lcl_burn0214 by analyzing its immune checkpoint gene expression and the ability of autologous tumor infiltrating lymphocytes (TIL_burn0214, TIL) to counteract lcl_burn0214 ex vivo. Results Establishment of lcl_burn0214 cell line The tumor sample, tum_burn0214, was obtained during the surgery of a 46-year-old Caucasian male diagnosed with microsatellite-stable, moderately differentiated (T4N0M0 G2) rectal adenocarcinoma at the Burnasyan Federal Medical Biophysical Center FMBA of Russia. Fresh tumor tissue was minced and digested with collagenase IV, and the resulting cell suspension was cultured. Over the following weeks, lymphocyte-like cell clusters formed in suspension. The resulting cell line, lcl_burn0214, has undergone 70 passages (approximately 180 divisions), consistently forming floating clusters. (Fig. 1A). The doubling time of the lcl_burn0214 cell line at passage 49 was determined to be 36 hours. Characterization of lcl_burn0214 cell line We first characterized the cell type, heterogeneity, authenticity, and patient origin of lcl_burn0214 cell line. Immunophenotyping revealed CD45+, CD19+, and CD20+ expression, with no CD3 detected (Fig. 1B). At the same time, epithelial, endothelial, and fibroblast markers were minimally expressed, confirming B cell identity (Fig. 1C). The B cell receptor clonotype diversity in lcl_burn0214 was an order of magnitude lower compared to the tum_burn0214 sample (Fig. 1D, Supplementary Table 1), indicating monoclonality in the lcl_burn0214. Due to low B cell infiltration in the tum_burn0214 (Supplementary Table 2 A–C), we were unable to detect any shared clonotypes between the two samples. HLA genotyping of tum_burn0214 and lcl_burn0214 samples, along with short tandem repeat profiling of lcl_burn0214, PBMC_burn0214, and TIL_burn0214 samples, confirmed their genetic relationship (Supplementary Table 3). Furthermore, no related cell lines were found among 8,808 human cell lines in the Cellosaurus database. Thus, lcl_burn0214 was an authentic nearly monoclonal B-cell line of patient origin. EBV status and tumorigenic potential of the lcl_burn0214 cell line B cell lines can arise from diverse origins, notably, the uncontrolled ex vivo proliferation of B lymphocytes is frequently linked to EBV infection, neoplastic transformation, or a combination of both [13]. Consequently, we investigated the EBV status and tumorigenic potential of the established lcl_burn0214 cell line. RNA-seq data detected EBV gene expression in the lcl_burn0214 sample but not in the tum_burn0214 sample (110,100 reads versus zero reads mapped to the EBV genome, respectively). Real-time PCR further confirmed the presence of EBV DNA in the lcl_burn0214 but not in tum_burn0214, TIL_burn0214, or PBMC_burn0214 samples (data not shown). Interestingly, previous studies have reported that EBV is not consistently detected in PBMCs of EBV-positive carriers or tumor samples from EBV-positive nasopharyngeal carcinoma patients [14, 15]. To exclude the possibility that lcl_burn0214 was infected with the laboratory EBV B95-8 strain, we looked at single nucleotide polymorphisms (SNPs) across the EBV reference genome. RNA-seq analysis of 10 B95-8_LCLs (Geuvadis consortium dataset) and 5 wild-type EBV infected bona fide B lymphoma cell lines (Quentmeier H. dataset) revealed SNP patterns in lcl_burn0214 and B lymphoma cell lines, differing from B95-8_LCLs, which, in turn, closely matched the reference genome (Fig. 2A). This indicates infection of the lcl_burn0214 cell line with wild-type EBV. As EBV-infected immortalized B cell lines are known to exhibit varying growth agility in immunodeficient mice [16] we evaluated the tumorigenic potential of the lcl_burn0214 cell line in NU–A/A Tyrc/Tyrc Foxn1nu/Foxn1nu mice. Initial growth was observed in four out of five mice, but all except one graft regressed over time. Histological analysis of the excised tumor revealed extensive necrosis with few viable blasts pointing at low tumorigenic potential of the cell line (supplementary Fig. 1 A-C). Gene expression analysis of the lcl_burn0214 cell line alongside lymphoblastoid and non-Hodgkin lymphoma cell lines To better position the lcl_burn0214 cell line within the lymphoblastoid–lymphoma spectrum we analyzed gene expression in the lcl_burn0214 cell line alongside cell lines from the Geuvadis and Quentmeier datasets. We selected RNA-seq data of 10 B95-8_LCLs, 4 EBV-positive and 5 EBV-negative B lymphomas from white males to minimize sex and race discrepancies (Supplementary Table 4). Principal component analysis and hierarchical clustering (Fig. 2B, C) revealed two major clusters. Notably, the lcl_burn0214 along with hairy cell leukemia (HCL) cell lines Bonna-12 and HC-1, and prolymphocytic leukemia (PLL) cell lines MEC-1 and PGA-1 clustered with B95-8_LCLs. In contrast, five EBV-negative lymphoma cell lines and one EBV-positive lymphoma cell line formed a separate cluster, indicating that clustering was independent of EBV infection, as exemplified by the PEL cell lines BC-3 and CRO-AP2 with differing EBV statuses. Collectively, our data show that the established lcl_burn0214 B cell line is lymphoblastoid, infected with a wild-type EBV strain, and exhibits limited tumorigenicity. The role of tumor-infiltrating B cells remains controversial, with studies supporting both pro- and anti-tumor effects [17]. EBV-transformed B cells further complicate this dynamic by adopting a regulatory B cell-like phenotype, promoting Treg cell development, and suppressing cytotoxic CD8 T cell proliferation and function through the PD-1/PD-L1 axis [18]. Accordingly, we subsequently explored the immunomodulating properties of the EBV-infected lcl_burn0214 B cell line by examining its immune checkpoint gene expression on the one hand, and the ability of TIL_burn0214 to counteract lcl_burn0214 B cell line ex vivo on the other. We compared checkpoint gene expression in lcl_burn0214, wild-type B lymphomas and B95-8_LCLs. Almost all samples showed low or negligible expression of HAVCR1 (TIM-1), TNFRSF14 (HVEM), IDO1, and HHLA2, but expressed TNFSF9 (4-1BB ligand), and CD70. Except for Hodgkin lymphoma cell line HDLM-2 cell lines split into two clusters, with lcl_burn0214, B95-8_LCLs, and HCL/PLL entities showing distinct immune checkpoint gene expression patterns compared to bona fide B lymphoma cell lines (Fig. 2D). The clusters differed in transcript levels for genes such as CD274 (PD-L1), TNFSF4 (OX40L), CSF1 and CD27, associated with positive and negative immune regulation. B cell contamination and specific anti-EBV activity of autologous tumor-infiltrating T lymphocytes TIL_burn0214 Finally, to assess the presence of EBV-specific clones among TIL_burn0214 as a marker of the TILs' ability to detect EBV-infected B cells, we conducted a cocultivation assay with lcl_burn0214. Reactivity to EBV-infected lcl_burn0214 was evaluated using IFN-γ ELISPOT assay, revealing that nearly 0.1% of total TILs were specific to EBV (Fig. 3A). Additionally, we evaluated whether TIL_burn0214 expanded from the rectal tumor specimen tum_burn0214 were contaminated with B cells, using immunophenotyping. The TILs were positive for CD45, CD3 markers but negative for the CD19, CD20 markers (Fig. 3B). As mentioned earlier, PCR analysis confirmed that TILs were EBV-negative. The results indicate no B cell contamination in the TIL_burn0214 culture and the presence of EBV-specific T cell clones, suggesting that TIL_burn0214 can counteract potential EBV-driven B cell proliferation during ex vivo culture. Discussion Studying EBV-immortalized B cell lines derived from tumor tissues during ex vivo culture and their interactions with TILs is of interest due to the expanding clinical use of TIL therapy. EBV-driven B cell outgrowth poses a potential risk during TIL expansion. Moreover, EBV-transformed B cells may modulate immune responses by altering T cell receptor repertoires, promoting regulatory T cell proliferation, and ultimately affecting TIL therapy efficacy—areas that remain underexplored [1,10,18]. Here, we describe the spontaneous emergence of a wild-type EBV-transformed B cell line, lcl_burn0214, from a rectal cancer specimen during ex vivo culture. The cell line formed lymphocyte-like clusters, grew in suspension, and underwent approximately 180 divisions. Immunophenotyping confirmed its B cell identity (CD45+, CD19+, CD20+, CD3–), with no expression of epithelial, endothelial, or fibroblast marker genes detected. A significant reduction in B cell receptor clonotype diversity relative to the parental tumor indicated monoclonality. Genetic authentication via HLA genotyping and STR profiling confirmed patient origin and excluded cross-contamination. SNP analysis identified infection with a wild-type EBV strain. Similar spontaneous immortalization of B cells during ex vivo tumor culture has been reported previously for ovarian, gastric, rectal tumors, leukaemia [4–6]. EBV-transformed B cells can proliferate unchecked in immunocompromised hosts [19]. TIL therapy typically involves immunosuppression, which may facilitate uncontrolled proliferation of EBV-transformed B cells in vivo [3], notably, if they contaminate the TIL product. However, the ability of B cell lines to proliferate and form tumors in immunocompromised mice, as well as B lymphocytic tumor formation in immunocompromised mice engrafted with tumor tissues, varies [7–10, 16]. In our study, lcl_burn0214 showed only transient, limited tumor growth in nude mice, with most grafts regressing and necrosis predominating in excised tissues of a single tumor, indicating low tumorigenicity. Other reports demonstrate that EBV-infected B cells and LCLs can form tumors in more severely immunocompromised mice (e.g., SCID, NOG) [7–10, 16]. Thus, lcl_burn0214’s tumorigenic potential in such models cannot be excluded. Immortalized B cell lines have been variably designated as lymphomas, lymphocytic tumors, or B lymphoblastoid cell lines by different authors, reflecting inconsistencies in their classification and origin [7–10]. To objectively evaluate the properties of our cell line, we performed a comparative gene expression analysis of the lcl_burn0214 line against lymphoblastoid cell lines and bona fide non-Hodgkin lymphoma cell lines. The analysis supported the lymphoblastoid nature of lcl_burn0214, as it clustered with LCLs and certain leukemia cell lines (e.g., hairy cell leukemia and prolymphocytic leukemia) rather than with B cell lymphomas. The clustering patterns aligned with previous reports questioning the authenticity of the leukemia cell lines Bonna-12 and HC-1, suggesting they may represent EBV-transformed bystander B cells rather than malignant clones [20,21]. Additionally, the origins of MEC-1 and PGA-1 cell lines are uncertain. This incidental finding underscores the importance of rigorous cell line authentication. On the one hand, EBV-infected B cells are recognized by T cells [22]; on the other hand, EBV is known to manipulate B cell metabolism. EBV-transformed immortalized B cells can exhibit immunomodulatory features that help them evade T cell–mediated immune responses. This is achieved, in part, by upregulating the expression of programmed cell death ligand-1 (PD-L1), which inhibits T cell function, and by downregulating the interferon response [1, 18]. We investigated the immunomodulatory features of the lcl_burn0214 cell line by profiling the expression of immune checkpoint genes in comparison with lymphoblastoid cell lines and bona fide B lymphoma cell lines. The lcl_burn0214 cell line, along with LCLs, displayed a distinct immune checkpoint gene expression signature compared to lymphoma cell lines. Of particular interest was the co-expression of CD70 and CD27, which is not observed on normal hematopoietic cells but is found on hematological malignancies and has been implicated in tumor progression according to previous studies [23]. In our study, CD70 and CD27 co-expression was detected on lymphoblastoid cells and the lcl_burn0214 cell line but not on bona fide B lymphoma cell lines. The expression of CD274 (PD-L1), which may reflect a regulatory B cell-like phenotype, was also detected in lcl_burn0214. This finding is consistent with previous reports demonstrating that EBV-transformed B cells can promote regulatory T cell development and suppress cytotoxic T cell responses via the PD-1/PD-L1 axis [1,18]. Additionally, the expression of colony-stimulating factor-1 (CSF-1) was observed in lcl_burn0214 cells. CSF-1 is known to recruit tumor-associated macrophages that possess immunosuppressive properties, thereby contributing to tumor immune evasion [24]. Interestingly, lcl_burn0214 also expressed TNFSF4 (OX40L), which has roles opposing those of CD274 and CSF-1 in immune regulation. TNFSF4 is a co-stimulatory molecule that enhances the activation and survival of CD4 + and CD8 + T cells [22, 25]. The combined gene expression of PD-L1, CSF-1, and OX40L in lcl_burn0214 cells may suggest a complex impact on the immune response. Despite the expression of immune checkpoint molecule genes by lcl_burn0214 cells, autologous tumor-infiltrating lymphocytes exhibited specific anti-EBV activity, demonstrated by interferon-gamma production in coculture assays with lcl-burn0214. Additionally, these TILs were both B cell- and EBV-negative. We did not examine the potential for lcl_burn0214 to skew T cell receptor (TCR) repertoires toward EBV antigens in vivo. Nevertheless, previous studies have demonstrated a shift in TCR repertoires toward EBV-specific clones, accompanied by repertoire narrowing, both in vivo and ex vivo, notably during the generation of T cell products targeting EBV upon cocultivation with autologous lymphoblastoid cell lines [10, 22]. In conclusion, we report an immortalized wild-type EBV-infected B cell line, lcl_burn0214 derived from a rectal tumor specimen, which exhibits a gene expression profile more similar to lymphoblastoid cells than to malignant B cell lymphomas. This cell line expresses several immune checkpoint molecules, including CD274, CSF-1 and TNFSF4. Meanwhile, autologous tumor-infiltrating lymphocytes demonstrate anti-EBV activity and are negative for both B cell contamination and EBV. Although not investigated here, EBV-infected B-TILs may promote regulatory T cell expansion or alter TCR repertoires, potentially impairing TIL therapy efficacy. Our findings underscore the importance of investigating the relationships between EBV-transformed B-TILs and autologous TILs, as well as screening TIL products for B cells and EBV during manufacturing, to optimize therapeutic outcomes. Materials and methods Patient information The patient was a 46-year-old Caucasian male diagnosed with microsatellite-stable, moderately differentiated (T4N0M0 G2) rectal adenocarcinoma. The tumor sample (designated tum_burn0214) and the blood sample were obtained during surgery. For tumor and blood samples from a patient this study was approved by the local institutional review board of the Burnasyan Federal Medical Biophysical Center FMBA of Russia under protocol RU-FMBC-23-07-2020. The study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from the patient prior to his participation. Tumor tissue and blood processing The tumor sample tum_burn0214 was placed in DMEM supplemented with 1× antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA USA) and maintained at 2–8°C until processing, which occurred within 2 hours of surgery. The sample was then cut into 1.5 × 1.5 mm fragments. Four fragments were used fresh to establish primary tumor cultures. A portion of the specimen was preserved in IntactRNA solution (Evrogen, Moscow, Russia) for subsequent RNA extraction, while the remaining fragments were cryopreserved in DMEM/F12 supplemented with 45% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) for tumor-infiltrating lymphocyte (TIL) expansion studies. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque density gradient centrifugation and cryopreserved in RPMI1640 containing 45% FBS and 10% DMSO. Primary Cell Line Establishment Four fresh tumor fragments, tum_burn0214 (1.5 × 1.5 mm each), were enzymatically digested with collagenase IV for 1 hour. After washing with phosphate-buffered saline (PBS), The resulting cell suspension was placed in a 6-well culture plate containing DMEM/F12 medium supplemented with 10% FBS, 2 mM Glutamax, and 1× antibiotic/antimycotic, and maintained in a humidified atmosphere with 5% CO₂. The culture medium was replaced weekly. When the cell density reached 1–1.5 million cells per milliliter (cells/mL), the cells were split to a concentration of 0.2–0.3 million cells/mL and transferred to new culture flasks. Cryopreservation of the established cell line, lcl_burn0214, was performed at passages 6, 15, 35, 46, and 62 using DMEM/F12 supplemented with 45% FBS and 10% DMSO. Thawing tests were successfully conducted for all specified passages. Tumor-Infiltrating Lymphocyte (TIL) TIL_burn0214 Expansion Thawed tumor fragments, tum_burn0214 (5–10 fragments per well of a 6-well plate), were placed in 2 mL of serum-free lymphocyte culture medium HIPP-T009 (Bioengine, Shanghai, China) supplemented with 5,000 units/mL IL-2 (Ronkoleukin, Biotech, Moscow, Russia) and 1× antibiotic/antimycotic (TIL culture medium). The ImmunoCult™ Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Vancouver, Canada) was added at a concentration of 25 µL/mL to activate T cells on day 0. Reactivation with the T Cell Activator was performed every 10–12 days in accordance with the manufacturer’s protocol. The TIL medium was refreshed two days post-activation. Upon reaching a density of 1–1.5 million cells/mL, TIL_burn0214 cells were subcultured into new flasks. Expanded TILs were subsequently characterized for phenotype and anti-EBV activity using the ELISPOT assay. In vivo Tumorigenicity The tumorigenic potential of lcl_burn0214 cells was evaluated in vivo by subcutaneous injection into five immunodeficient male NU–A/A Tyrc/Tyrc Foxn1nu/Foxn1nu mice (BALB/c nude strain, 6–8 weeks old; obtained from the Laboratory Animals Breeding Facility BIBCh, Pushchino, Russia). Animals were maintained under specific pathogen-free conditions in the Vivarium of the Department of Experimental Biology at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (IBCh RAS). Animal procedures were approved by the local institutional review board of the Vivarium of the Department of Experimental Biology at the IBCh RAS under protocol #378/2023 and carried out in accordance with the institutional guidelines. Lcl_burn0214 cells at passage 14 were harvested and resuspended in phosphate-buffered saline at a concentration of 1×10 7 cells/mL. Each mouse received a subcutaneous injection of 0.1 mL cell suspension (total 1x10 6 cells) into the right flank. Tumor growth was monitored weekly by measuring two orthogonal diameters with an electronic caliper. Mice were euthanized 75 days post-injection. The only tumor that reached 200 mm³ was excised, fixed in 4% neutral-buffered formaldehyde, processed for paraffin embedding. Tissue sections (3.5 mkm) were prepared for hematoxylin and eosin staining. Short tandem repeats (STR) analysis TIL_burn0214, PBMC_burn0214, the lcl_burn0214 cell line at passages 15, 53, from the patient were subjected to STR analysis. 27 loci were amplified using PowerPlex® Fusion 6C System STR and detected by the Applied Biosystems 3500 Series Genetic Analyzer. Data were analyzed with GeneMapper ID‑X Software v1.6. The STR profile of the lcl_burn0214 cell line was compared with the Cellosaurus database to identify any related cell lines using the CLASTR 1.4.4 online tool [26]. Flow cytometry Phenotypic analysis was performed on lcl_burn0214 and TIL_burn0214 cell cultures using the following monoclonal antibodies purchased from Sony Biotechnology, San Jose, Ca, USA: CD3_FITC (2186530), CD19_APC (2111055), and CD45_FITC (2120025), CD4_APC_cy7, CD8_PE (2105255), CD20_PE (2111525). DAPI was used to counterstain live cells. Cells (1x10 5 ) were washed in PBS and resuspended in a staining buffer containing the appropriate antibodies. Surface marker staining was performed for 20 min at 4 °C. After incubation, cells were washed twice, collected in 100 μl of PBS, and immediately analyzed using the ACEA NovoCyte® flow cytometer VBR system (Agilent Technologies, Santa Clara, CA, USA). NovoCyte Express software was used for plot analysis. Elispot assay To evaluate the functional response of tumor-infiltrating lymphocytes (TILs) to the EBV-positive autologous lcl_burn0214 cell line, co-culture experiments were performed. The TIL_burn0214 and lcl_burn0214 cell line were co-cultured at effector-to-target (E:T) ratios of 1:1 and 1:10 in duplicate wells of 96-well flat-bottom plates pre-coated with anti-IFN-γ antibodies. Each well contained either 5 000 or 50 000 TIL_burn0214 cells and 50 000 lcl_burn0214 cells. TIL_burn0214 cells were thawed and used immediately in the assay. Two independent vials of TIL_burn0214 were utilized as biological replicates. After 18 hours of incubation, IFNγ production was quantified using a commercially available ELISpot kit (ImmunoSpot® ELISpot kit, Cellular Technology Limited, C.T.L., Cleveland, OH, USA), following the manufacturer’s protocol. Negative controls included wells containing either 50 000 lcl_burn0214 or 50 000 TIL_burn0214 cultured alone. Developed ELISpot plates were analyzed using the Immunospot analyzer software (C.T.L.), and the mean number of IFN-γ spot-forming units (SFUs) was calculated for each condition: TIL_burn0214 co-cultured with lcl_burn0214, TIL_burn0214 alone, and lcl_burn0214 alone. RNA extraction and construction of transcriptome libraries RNA extraction from tumor tissue tum_burn0214 was performed using the All Prep RNA/DNA Mini Kit (Qiagen, Hilden, Germany). Tissue fragments preserved in Intact RNA solution were minced on a sterile Perti dish until they were as small as possible. Tissue fragments were then transferred to a 2 ml tube containing three stainless steel spheres of 4.8 mm diameter, to which 700 mcl of RLT lysis solution containing 1% beta-mercaptoethanol was added. Tissue fragments were homogenized using the Tissue Lyser instrument (Qiagen, Hilden, Germany) under the following conditions: 1 minute, 25/s. The next steps of RNA extraction were performed according to the manufacturer's instructions for animal tissues. RNA from lcl_burn0214 was also isolated using the All Prep RNA/DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions for animal cells. Approximately 4*10 6 fresh pelleted cells were used as input. Homogenization was performed using a 20G needle with a syringe. Total RNA integrity number (RIN) was determined using RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA) with Bioanalyzer 2100 instrument (Agilent, USA). TURBO DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to remove DNA traces. For the construction of the transcriptome library, 500 ng of total RNA was collected. First, RNA was enriched for poly(A) fraction using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA). Next, non-directional transcriptome libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, USA) with the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (New England Biolabs, USA) according to the manufacturer's instructions. Paired-end libraries were sequenced on the Illumina NovaSeq 6000 instrument at 2 × 100 cycles (Illumina, San Diego, CA, USA). Bioinformatics pipeline Data preparation for analysis Quality control by FastQC (0.12.1) [27] and MultiQC (1.15) [28] was performed before and after adapter trimming by cutadapt (version 4.4) [29] and quality filtering by trimmomatic (version 0.39) [30] for the whole data set. Pseudoalignment was performed by salmon (1.10.2) [31] for Gencode.v43 transcriptome with full genome decoy index. Summarizing for gene was performed by tximport (1.26.1) [32] using the length scaled TPM metrics and ignoring transcripts version. Expression and dispersion for genes and transcripts has been estimated by edgeR (version 3.40.2) [33]. For the further analysis CPM data from edgeR was used with filtering by maximal mean in any group > 4 CPM. PCA analysis was realized by PCAtools (v2.10.0) [34] on log2 transformed CPM removing genes with the lowest 10% variance. Plots were plotted with ggplot2 (v3.4.3) [35]. HLA Typing for RNA-seq data (tum_burn0214 and lcl_burn0214 samples) HLA typing was performed with HLA-HD [36] against IMGT/HLA database (version 3.50) [37] using RNA-seq data from tum_burn0214 and lcl_burn0214 samples. EBV reference genome aligning RNA-seq reads were aligned to the human reference genome using STAR version 2.7.10b [38] in 2-pass mode. The reference genome used was the GRCh38 (hg38) assembly provided by the GDC [39], which includes decoy and 200 viral genomes including the EBV; the genome index for STAR was generated using GENCODE v43 gene annotations. Analysis of public cancer cell line transcriptomics data Lymphoma cell line transcriptomic data (Quentmeier H. dataset) [40], were downloaded from ENA (PRJEB30312) [41], B95-8 EBV strain-infected lymphoblastoid B cell lines (Geuvadis consortium dataset) [42] were downloaded from IGSR [43]. cell type deconvolution analysis Deconvolution of cell types were realized by MSP, xCell and EPIC methods using the immunedeconv R package (v2.1.3) [44]. BCR profiling analysis BCR repertoire were evaluated by MiXCR (version 4.6.0) [45] Declarations Data availability RNA-seq data produced in this study have been deposited at the BioProject and are available under accession number PRJNA1266483 Acknowledgements The authors thank core facilities of the Lopukhin FRCC PCM FMBA of Russia “Genomics, proteomics, metabolomics” () for performing sequencing The authors thank Julia A. Bespyatykh, the head of Laboratory of Molecular Medicine, Lopukhin FRCC PCM FMBA of Russia for EBV detection technical support The authors thank Marina S. Krasilshchikova the head of the Vivarium of the Department of Experimental Biology at the IBCh RAS for technical support with animal model experiments The authors thank Anna Khalturina and LLC Impulstest for performing STR analysis Funding This work was supported by the «T-kletki» project funding 123032900030-7 by Federal Medical Biological Agency Author information Authors and Affiliations Lopukhin FRCC PCM FMBA of Russia, Moscow, Russian Federation Tatiana V. Petrova, Daria V. Kuznetzova, Alexandra V. Kanygina, Liubov O. Skorodumova, Elena I. Sharova, Viktor A. Ivanov The Burnasyan Federal Medical Biophysical Center FMBA of Russia Tatiana A. Astrelina, Svetlana E. Varlamova Contributions TVP EIS designed the study, TVP, DVK, LOS, VAI, EIS performed and interpreted experimental data, EIS, AVK, TVP performed and interpreted bioinformatic analysis, TAA, SEV provided tumor specimen and clinical data, TVP wrote the original draft with contributions from all authors. All authors edited the manuscript. The final version of the manuscript was approved by all authors. Corresponding authors Correspondence to Tatiana V. Petrova [email protected] , Elena I. Sharova [email protected] Additional Information The authors declare no competing interests. Ethics declarations For tumor and blood samples from a patient this study was approved by the local institutional review board of the Burnasyan Federal Medical Biophysical Center FMBA of Russia under protocol RU-FMBC-23-07-2020. The study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from the patient prior to his participation. Animal procedures were approved by the local institutional review board of the Vivarium of the Department of Experimental Biology at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (IBCh RAS) under protocol #378/2023 and carried out in accordance with the institutional guidelines. References Sausen, D. G., Poirier, M. C., Spiers, L. M., Smith, E. N. Mechanisms of T cell evasion by Epstein-Barr virus and implications for tumor survival. Frontiers in Immunology 14 , 1289313; 10.3389/fimmu.2023.1289313 (2023). Lareau, C. A. et al. Latent human herpesvirus 6 is reactivated in CAR T cells. Nature 623 , 608–615 (2023). Rohaan, M. W. et al. Tumor-infiltrating lymphocyte therapy or ipilimumab in advanced melanoma. New England Journal of Medicine 387 , 2113–2125 (2022). Zhang, L. et al. Characterization of latently infected EBV+ antibody-secreting B cells isolated from ovarian tumors and malignant ascites. 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Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research 4 , 1521–1521 (2016). McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40 , 4288–4297 (2012). Blighe, K. PCAtools: everything Principal Component Analysis. Bioconductor.org https://www.bioconductor.org/packages/devel/bioc/vignettes/PCAtools/inst/doc/PCAtools.html (2025). Wickham, H. ggplot2. SpringerLink 10.1007-978-3-319-24277-4 (2016). Kawaguchi, S., Koichiro Higasa, Shimizu, M., Yamada, R. & Matsuda, F. HLA‐HD: An accurate HLA typing algorithm for next‐generation sequencing data. Human Mutation 38 , 788–797 (2017). Robinson, J. et al. The IPD and IMGT/HLA database: allele variant databases. Nucleic Acids Research 43 , D423–D431 (2014). Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2012). GDC Reference Files | NCI Genomic Data Commons. Cancer.gov https://gdc.cancer.gov/about-data/gdc-data-processing/gdc-reference-files (2025). Quentmeier, H. et al. The LL-100 panel: 100 cell lines for blood cancer studies. Scientific Reports 9 , (2019). EMBL-EBI. ENA Browser. Ebi.ac.uk https://www.ebi.ac.uk/ena/browser/home (2025). Lappalainen, T. et al. Transcriptome and genome sequencing uncovers functional variation in humans. Nature 501 , 506–511 (2013). Data portal | 1000 Genomes. Internationalgenome.org https://www.internationalgenome.org/data-portal/sample (2025). Sturm, G., Finotello, F. & List, M. Immunedeconv: An R Package for Unified Access to Computational Methods for Estimating Immune Cell Fractions from Bulk RNA-Sequencing Data. Methods in Molecular Biology 223–232; 10.1007/978-1-0716-0327-7_16 (2020). Bolotin, D. A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nature Methods 12 , 380–381 (2015). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Electronic supplementary material Supplementary Figure 1 Supplementary Tables 1-4 Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 27 Jun, 2025 Reviewers agreed at journal 19 Jun, 2025 Reviewers invited by journal 17 Jun, 2025 Editor assigned by journal 17 Jun, 2025 Editor invited by journal 12 Jun, 2025 Submission checks completed at journal 11 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6835907","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472711621,"identity":"4a4b576e-65fd-4616-b8fc-8f3d83bb27f6","order_by":0,"name":"Tatiana V. 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Surface expression of CD45, CD19, CD20, CD3 markers in lcl_burn0214 cell line. Cells were counterstained with DAPI to exclude dead cells from analysis. Isotype controls are colored black on histograms, CD45, CD3 markers are colored red on histograms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Selected epithelial, endothelial, fibroblast and hematopoietic expression markers in lcl_burn0214 and tum_burn0214 samples. Bars represent gene expression level in CPM (count per million).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Number of clonotypes for BCR chains identified in tum_burn0214 and lcl_burn0214 samples. Bars represent the number of IGH, IGK and IGL clones\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6835907/v1/43048fa0b60792ff653ab6e3.png"},{"id":85171337,"identity":"ea5032da-5040-43d0-b4c7-546519c02ee7","added_by":"auto","created_at":"2025-06-23 05:42:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBulk RNA-seq data analysis of lcl_burn0214, 10 B95-8_LCLs, 5 EBV+ and 5 EBV- B cell lymphoma cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Hierarchical clustering of the SNP sites across the EBV genome extracted from Rna-seq data of 10 B95-8_LCLs from Geuvadis consortium dataset, 5 EBV+lymphoma cell lines from Quentmeier H. dataset and spontaneous lcl_burn0214 cell line. Each row in the heatmap represents an SNP position, and each column represents a cell line. Reference alleles are colored dark blue, while non-reference alleles are colored light blue. The reference genome sequence is the type 1 EBV surrogate genome sequence (NC_007605), constructed based on the B95-8 and Raji EBV genomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Similarity of malignant and non-malignant B cell lines by principal component analysis of the following samples: B95-8_LCLs, lcl_burn0214, EBV-negative and EBV-positive lymphomas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Similarity of malignant and non-malignant B cell lines by hierarchical clustering of transcriptome correlation of the following samples: B95-8_LCLs, lcl_burn0214, EBV-negative and EBV-positive lymphomas. The correlation plot is symmetric on one diagonal. Correlation is pointed by color the more red the greater correlation between the two samples. Cell lines are color-coded according to their cellular origin: B_NHL, green; HL, orange; B95-8_LCLs, blue; lcl_burn0214, violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Hierarchical clustering of the expression level of 28 immune checkpoint genes of 10 B95-8_LCLs, 5 EBV-negative, 5 EBV-positive lymphomas and lcl_burn0214 cell line. Each row represents an immune checkpoint gene, each column represents a sample.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6835907/v1/2882fd0b9f8baa775b1d41ed.png"},{"id":85172116,"identity":"323855b4-3c0b-47f6-8cc9-0cd0519bc158","added_by":"auto","created_at":"2025-06-23 05:50:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of TIL_burn0214.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Frequency of TIL_burn0214 cells producing interferon γ (IFN-γ) in response to coculture with lcl_burn0214. Bars represent the mean number of IFN-γ spots per 1 mln of TIL_burn0214 or lcl_burn0214 (when TIL_0214 are not added to cell culture). Two vials of TIL_burn0214 were used in co-culture assay as independent repeats. TIL_burn0214 cells and the lcl_burn0214 cell line were co-cultured at effector-to-target (E:T) ratios of 1:1 and 1:10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Flow cytometry dot plot showing positive staining of TIL_burn0214 with CD45, CD3, markers and negative staining with CD19, CD20 markers. Cells were counterstained with DAPI to exclude dead cells from analysis. Isotype controls are colored black on histograms, CD45, CD3, CD19, CD20 markers are colored red on histograms.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6835907/v1/47b932ef6179b709d3d0db16.png"},{"id":97178467,"identity":"e2e8e9e3-35a8-4683-9fb2-192f3b6418af","added_by":"auto","created_at":"2025-12-01 16:10:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1456566,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835907/v1/1227a434-7004-4440-a694-85c4bff04684.pdf"},{"id":85172115,"identity":"d6e3e810-ffac-4a3d-95a6-9fff2ebd5bab","added_by":"auto","created_at":"2025-06-23 05:50:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":213111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectronic supplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 1\u003c/p\u003e\n\u003cp\u003eSupplementary Tables 1-4\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6835907/v1/99b93b02eb5db61a22c02d46.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSpontaneous EBV-positive B cell line from rectal tumor: characterization, interaction with autologous tumor-infiltrating T lymphocytes, comparison with B lymphoma cell lines\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEBV, a member of the herpesvirus family, primarily infects B lymphocytes and persists as an episome throughout the host's lifespan, evading T cell-mediated immune responses. EBV is associated with B cell lymphomas and can trigger post-transplant lymphoproliferative disease in immunocompromised individuals [1].\u003c/p\u003e \u003cp\u003eRecent studies have raised concerns about herpesvirus-associated risks in T cell therapies, including chimeric antigen receptor (CAR) T cell and tumor-infiltrating T lymphocyte treatments. Latent human herpesvirus 6 reactivation has been demonstrated in CAR-T cells in vivo, along with a reported case of EBV-positive B lymphoma following TIL therapy for metastatic melanoma [2, 3]. Previous studies have reported the spontaneous ex vivo outgrowth of immortalized EBV-transformed B cell lines from various solid and hematological tumors, including ovarian cancer, colorectal cancer, and leukemia [4\u0026ndash;6]. Several animal studies have demonstrated that highly immunocompromised mice engrafted with patient tumor tissues\u0026mdash;including ovarian, gastric, breast, lung, and colorectal tumors\u0026mdash;often develop metastatic EBV-positive B cell lymphocytic tumors rather than patient-derived epithelial tumor xenografts [7\u0026ndash;10]. Tumor fragments used to generate cell products for tumor-infiltrating lymphocyte (TIL) therapy contain diverse cell populations, such as TILs and tumor-infiltrating B lymphocytes (B-TILs) [11]. Therefore, during ex vivo TIL expansion for cell therapy, there is a risk that EBV-transformed B-TILs may proliferate concurrently, particularly in colorectal cancer, where EBV-positive lymphocyte infiltration of the tumor occurs in approximately 40% of cases [12]. Also EBV-transformed B cells have the potential to modulate immune responses by reshaping T cell receptor repertoire [10]. These features of EBV-transformed B-TILs may significantly influence the efficacy and safety of TIL therapies, but remain insufficiently explored.\u003c/p\u003e \u003cp\u003eIn this study, we characterized a wild-type EBV-transformed B cell line, lcl_burn0214, derived spontaneously from a rectal cancer specimen during ex vivo culture. Using RNA-seq, we compared its gene expression with established EBV-positive and EBV-negative B lymphoma lines and B95-8 EBV-infected lymphoblastoid cell lines (LCLs, B95-8_LCLs). We then assessed possible immune regulating features of lcl_burn0214 by analyzing its immune checkpoint gene expression and the ability of autologous tumor infiltrating lymphocytes (TIL_burn0214, TIL) to counteract lcl_burn0214 ex vivo.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eEstablishment of lcl_burn0214 cell line\u003c/h2\u003e\n\u003cp\u003eThe tumor sample, tum_burn0214, was obtained during the surgery of a 46-year-old Caucasian male diagnosed with microsatellite-stable, moderately differentiated (T4N0M0 G2) rectal adenocarcinoma at the Burnasyan Federal Medical Biophysical Center FMBA of Russia.\u003c/p\u003e\n\u003cp\u003eFresh tumor tissue was minced and digested with collagenase IV, and the resulting cell suspension was cultured. Over the following weeks, lymphocyte-like cell clusters formed in suspension. The resulting cell line, lcl_burn0214, has undergone 70 passages (approximately 180 divisions), consistently forming floating clusters. (Fig. 1A). The doubling time of the lcl_burn0214 cell line at passage 49 was determined to be 36 hours.\u003c/p\u003e\n\u003ch2\u003eCharacterization of lcl_burn0214 cell line\u003c/h2\u003e\n\u003cp\u003eWe first characterized the cell type, heterogeneity, authenticity, and patient origin of lcl_burn0214 cell line. Immunophenotyping revealed CD45+, CD19+, and CD20+ expression, with no CD3 detected (Fig. 1B). At the same time, epithelial, endothelial, and fibroblast markers were minimally expressed, confirming B cell identity (Fig. 1C). The B cell receptor clonotype diversity in lcl_burn0214 was an order of magnitude lower compared to the tum_burn0214 sample (Fig. 1D, Supplementary Table 1), indicating monoclonality in the lcl_burn0214. Due to low B cell infiltration in the tum_burn0214 (Supplementary Table 2 A\u0026ndash;C), we were unable to detect any shared clonotypes between the two samples.\u003c/p\u003e\n\u003cp\u003eHLA genotyping of tum_burn0214 and lcl_burn0214 samples, along with short tandem repeat profiling of lcl_burn0214, PBMC_burn0214, and TIL_burn0214 samples, confirmed their genetic relationship (Supplementary Table 3). Furthermore, no related cell lines were found among 8,808 human cell lines in the Cellosaurus database.\u003c/p\u003e\n\u003cp\u003eThus, lcl_burn0214 was an authentic nearly monoclonal B-cell line of patient origin.\u003c/p\u003e\n\u003ch2\u003eEBV status and tumorigenic potential of the lcl_burn0214 cell line\u003c/h2\u003e\n\u003cp\u003eB cell lines can arise from diverse origins, notably, the uncontrolled ex vivo proliferation of B lymphocytes is frequently linked to EBV infection, neoplastic transformation, or a combination of both [13]. Consequently, we investigated the EBV status and tumorigenic potential of the established lcl_burn0214 cell line. RNA-seq data detected EBV gene expression in the lcl_burn0214 sample but not in the tum_burn0214 sample (110,100 reads versus zero reads mapped to the EBV genome, respectively). Real-time PCR further confirmed the presence of EBV DNA in the lcl_burn0214 but not in tum_burn0214, TIL_burn0214, or PBMC_burn0214 samples (data not shown). Interestingly, previous studies have reported that EBV is not consistently detected in PBMCs of EBV-positive carriers or tumor samples from EBV-positive nasopharyngeal carcinoma patients [14, 15].\u003c/p\u003e\n\u003cp\u003eTo exclude the possibility that lcl_burn0214 was infected with the laboratory EBV B95-8 strain, we looked at single nucleotide polymorphisms (SNPs) across the EBV reference genome. RNA-seq analysis of 10 B95-8_LCLs (Geuvadis consortium dataset) and 5 wild-type EBV infected bona fide B lymphoma cell lines (Quentmeier H. dataset) revealed SNP patterns in lcl_burn0214 and B lymphoma cell lines, differing from B95-8_LCLs, which, in turn, closely matched the reference genome (Fig. 2A). This indicates infection of the lcl_burn0214 cell line with wild-type EBV.\u003c/p\u003e\n\u003cp\u003eAs EBV-infected immortalized B cell lines are known to exhibit varying growth agility in immunodeficient mice [16] we evaluated the tumorigenic potential of the lcl_burn0214 cell line in NU\u0026ndash;A/A Tyrc/Tyrc Foxn1nu/Foxn1nu mice. Initial growth was observed in four out of five mice, but all except one graft regressed over time. Histological analysis of the excised tumor revealed extensive necrosis with few viable blasts pointing at low tumorigenic potential of the cell line (supplementary Fig. 1 A-C).\u003c/p\u003e\n\u003ch2\u003eGene expression analysis of the lcl_burn0214 cell line alongside lymphoblastoid and non-Hodgkin lymphoma cell lines\u003c/h2\u003e\n\u003cp\u003eTo better position the lcl_burn0214 cell line within the lymphoblastoid\u0026ndash;lymphoma spectrum we analyzed gene expression in the lcl_burn0214 cell line alongside cell lines from the Geuvadis and Quentmeier datasets.\u003c/p\u003e\n\u003cp\u003eWe selected RNA-seq data of 10 B95-8_LCLs, 4 EBV-positive and 5 EBV-negative B lymphomas from white males to minimize sex and race discrepancies (Supplementary Table 4). Principal component analysis and hierarchical clustering (Fig. 2B, C) revealed two major clusters. Notably, the lcl_burn0214 along with hairy cell leukemia (HCL) cell lines Bonna-12 and HC-1, and prolymphocytic leukemia (PLL) cell lines MEC-1 and PGA-1 clustered with B95-8_LCLs. In contrast, five EBV-negative lymphoma cell lines and one EBV-positive lymphoma cell line formed a separate cluster, indicating that clustering was independent of EBV infection, as exemplified by the PEL cell lines BC-3 and CRO-AP2 with differing EBV statuses.\u003c/p\u003e\n\u003cp\u003eCollectively, our data show that the established lcl_burn0214 B cell line is lymphoblastoid, infected with a wild-type EBV strain, and exhibits limited tumorigenicity.\u003c/p\u003e\n\u003cp\u003eThe role of tumor-infiltrating B cells remains controversial, with studies supporting both pro- and anti-tumor effects [17]. \u0026nbsp;EBV-transformed B cells further complicate this dynamic by adopting a regulatory B cell-like phenotype, promoting Treg cell development, and suppressing cytotoxic CD8 T cell proliferation and function through the PD-1/PD-L1 axis \u0026nbsp;[18].\u003c/p\u003e\n\u003cp\u003eAccordingly, we subsequently explored the immunomodulating properties of the EBV-infected lcl_burn0214 B cell line by examining its immune checkpoint gene expression on the one hand, and the ability of TIL_burn0214 to counteract lcl_burn0214 B cell line ex vivo on the other.\u003c/p\u003e\n\u003cp\u003eWe compared checkpoint gene expression in lcl_burn0214, wild-type B lymphomas and B95-8_LCLs. Almost all samples showed low or negligible expression of HAVCR1 (TIM-1), TNFRSF14 (HVEM), IDO1, and HHLA2, but expressed TNFSF9 (4-1BB ligand), and CD70. Except for Hodgkin lymphoma cell line HDLM-2 cell lines split into two clusters, with lcl_burn0214, B95-8_LCLs, and HCL/PLL entities showing distinct immune checkpoint gene expression patterns compared to bona fide B lymphoma cell lines (Fig. 2D). The clusters differed in transcript levels for genes such as CD274 (PD-L1), TNFSF4 (OX40L), CSF1 and CD27, associated with positive and negative immune regulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eB cell contamination and specific anti-EBV activity of autologous tumor-infiltrating T lymphocytes TIL_burn0214\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eFinally, to assess the presence of EBV-specific clones among TIL_burn0214 as a marker of the TILs\u0026apos; ability to detect EBV-infected B cells, we conducted a cocultivation assay with lcl_burn0214. Reactivity to EBV-infected lcl_burn0214 was evaluated using IFN-\u0026gamma; ELISPOT assay, revealing that nearly 0.1% of total TILs were specific to EBV (Fig. 3A).\u003c/p\u003e\n\u003cp\u003eAdditionally, we evaluated whether TIL_burn0214 expanded from the rectal tumor specimen tum_burn0214 were contaminated with B cells, using immunophenotyping. The TILs were positive for CD45, CD3 markers but negative for the CD19, CD20 markers (Fig. 3B). As mentioned earlier, PCR analysis confirmed that TILs were EBV-negative. The results indicate no B cell contamination in the TIL_burn0214 culture and the presence of EBV-specific T cell clones, suggesting that TIL_burn0214 can counteract potential EBV-driven B cell proliferation during ex vivo culture.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudying EBV-immortalized B cell lines derived from tumor tissues during ex vivo culture and their interactions with TILs is of interest due to the expanding clinical use of TIL therapy. EBV-driven B cell outgrowth poses a potential risk during TIL expansion. Moreover, EBV-transformed B cells may modulate immune responses by altering T cell receptor repertoires, promoting regulatory T cell proliferation, and ultimately affecting TIL therapy efficacy\u0026mdash;areas that remain underexplored [1,10,18].\u003c/p\u003e \u003cp\u003eHere, we describe the spontaneous emergence of a wild-type EBV-transformed B cell line, lcl_burn0214, from a rectal cancer specimen during ex vivo culture. The cell line formed lymphocyte-like clusters, grew in suspension, and underwent approximately 180 divisions. Immunophenotyping confirmed its B cell identity (CD45+, CD19+, CD20+, CD3\u0026ndash;), with no expression of epithelial, endothelial, or fibroblast marker genes detected. A significant reduction in B cell receptor clonotype diversity relative to the parental tumor indicated monoclonality. Genetic authentication via HLA genotyping and STR profiling confirmed patient origin and excluded cross-contamination. SNP analysis identified infection with a wild-type EBV strain.\u003c/p\u003e \u003cp\u003eSimilar spontaneous immortalization of B cells during ex vivo tumor culture has been reported previously for ovarian, gastric, rectal tumors, leukaemia [4\u0026ndash;6].\u003c/p\u003e \u003cp\u003eEBV-transformed B cells can proliferate unchecked in immunocompromised hosts [19]. TIL therapy typically involves immunosuppression, which may facilitate uncontrolled proliferation of EBV-transformed B cells in vivo [3], notably, if they contaminate the TIL product. However, the ability of B cell lines to proliferate and form tumors in immunocompromised mice, as well as B lymphocytic tumor formation in immunocompromised mice engrafted with tumor tissues, varies [7\u0026ndash;10, 16]. In our study, lcl_burn0214 showed only transient, limited tumor growth in nude mice, with most grafts regressing and necrosis predominating in excised tissues of a single tumor, indicating low tumorigenicity. Other reports demonstrate that EBV-infected B cells and LCLs can form tumors in more severely immunocompromised mice (e.g., SCID, NOG) [7\u0026ndash;10, 16]. Thus, lcl_burn0214\u0026rsquo;s tumorigenic potential in such models cannot be excluded.\u003c/p\u003e \u003cp\u003eImmortalized B cell lines have been variably designated as lymphomas, lymphocytic tumors, or B lymphoblastoid cell lines by different authors, reflecting inconsistencies in their classification and origin [7\u0026ndash;10]. To objectively evaluate the properties of our cell line, we performed a comparative gene expression analysis of the lcl_burn0214 line against lymphoblastoid cell lines and bona fide non-Hodgkin lymphoma cell lines. The analysis supported the lymphoblastoid nature of lcl_burn0214, as it clustered with LCLs and certain leukemia cell lines (e.g., hairy cell leukemia and prolymphocytic leukemia) rather than with B cell lymphomas. The clustering patterns aligned with previous reports questioning the authenticity of the leukemia cell lines Bonna-12 and HC-1, suggesting they may represent EBV-transformed bystander B cells rather than malignant clones [20,21]. Additionally, the origins of MEC-1 and PGA-1 cell lines are uncertain. This incidental finding underscores the importance of rigorous cell line authentication.\u003c/p\u003e \u003cp\u003eOn the one hand, EBV-infected B cells are recognized by T cells [22]; on the other hand, EBV is known to manipulate B cell metabolism. EBV-transformed immortalized B cells can exhibit immunomodulatory features that help them evade T cell\u0026ndash;mediated immune responses. This is achieved, in part, by upregulating the expression of programmed cell death ligand-1 (PD-L1), which inhibits T cell function, and by downregulating the interferon response [1, 18].\u003c/p\u003e \u003cp\u003eWe investigated the immunomodulatory features of the lcl_burn0214 cell line by profiling the expression of immune checkpoint genes in comparison with lymphoblastoid cell lines and bona fide B lymphoma cell lines. The lcl_burn0214 cell line, along with LCLs, displayed a distinct immune checkpoint gene expression signature compared to lymphoma cell lines.\u003c/p\u003e \u003cp\u003eOf particular interest was the co-expression of CD70 and CD27, which is not observed on normal hematopoietic cells but is found on hematological malignancies and has been implicated in tumor progression according to previous studies [23]. In our study, CD70 and CD27 co-expression was detected on lymphoblastoid cells and the lcl_burn0214 cell line but not on bona fide B lymphoma cell lines.\u003c/p\u003e \u003cp\u003eThe expression of CD274 (PD-L1), which may reflect a regulatory B cell-like phenotype, was also detected in lcl_burn0214. This finding is consistent with previous reports demonstrating that EBV-transformed B cells can promote regulatory T cell development and suppress cytotoxic T cell responses via the PD-1/PD-L1 axis [1,18].\u003c/p\u003e \u003cp\u003eAdditionally, the expression of colony-stimulating factor-1 (CSF-1) was observed in lcl_burn0214 cells. CSF-1 is known to recruit tumor-associated macrophages that possess immunosuppressive properties, thereby contributing to tumor immune evasion [24]. Interestingly, lcl_burn0214 also expressed TNFSF4 (OX40L), which has roles opposing those of CD274 and CSF-1 in immune regulation. TNFSF4 is a co-stimulatory molecule that enhances the activation and survival of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells [22, 25].\u003c/p\u003e \u003cp\u003eThe combined gene expression of PD-L1, CSF-1, and OX40L in lcl_burn0214 cells may suggest a complex impact on the immune response.\u003c/p\u003e \u003cp\u003eDespite the expression of immune checkpoint molecule genes by lcl_burn0214 cells, autologous tumor-infiltrating lymphocytes exhibited specific anti-EBV activity, demonstrated by interferon-gamma production in coculture assays with lcl-burn0214. Additionally, these TILs were both B cell- and EBV-negative. We did not examine the potential for lcl_burn0214 to skew T cell receptor (TCR) repertoires toward EBV antigens in vivo. Nevertheless, previous studies have demonstrated a shift in TCR repertoires toward EBV-specific clones, accompanied by repertoire narrowing, both in vivo and ex vivo, notably during the generation of T cell products targeting EBV upon cocultivation with autologous lymphoblastoid cell lines [10, 22].\u003c/p\u003e \u003cp\u003eIn conclusion, we report an immortalized wild-type EBV-infected B cell line, lcl_burn0214 derived from a rectal tumor specimen, which exhibits a gene expression profile more similar to lymphoblastoid cells than to malignant B cell lymphomas. This cell line expresses several immune checkpoint molecules, including CD274, CSF-1 and TNFSF4. Meanwhile, autologous tumor-infiltrating lymphocytes demonstrate anti-EBV activity and are negative for both B cell contamination and EBV. Although not investigated here, EBV-infected B-TILs may promote regulatory T cell expansion or alter TCR repertoires, potentially impairing TIL therapy efficacy. Our findings underscore the importance of investigating the relationships between EBV-transformed B-TILs and autologous TILs, as well as screening TIL products for B cells and EBV during manufacturing, to optimize therapeutic outcomes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003e\u003cstrong\u003ePatient information\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe patient was a 46-year-old Caucasian male diagnosed with microsatellite-stable, moderately differentiated (T4N0M0 G2) rectal adenocarcinoma. The tumor sample (designated tum_burn0214) and the blood sample were obtained during surgery. For tumor and blood samples from a patient this study was approved by the local institutional review board of the Burnasyan Federal Medical Biophysical Center FMBA of Russia under protocol RU-FMBC-23-07-2020. The study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from the patient prior to his participation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eTumor tissue and blood processing\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumor sample tum_burn0214 was placed in DMEM supplemented with 1\u0026times; antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA USA) and maintained at 2\u0026ndash;8\u0026deg;C until processing, which occurred within 2 hours of surgery. The sample was then cut into 1.5 \u0026times; 1.5 mm fragments. Four fragments were used fresh to establish primary tumor cultures. A portion of the specimen was preserved in IntactRNA solution (Evrogen, Moscow, Russia) for subsequent RNA extraction, while the remaining fragments were cryopreserved in DMEM/F12 supplemented with 45% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) for tumor-infiltrating lymphocyte (TIL) expansion studies. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque density gradient centrifugation and cryopreserved in RPMI1640 containing 45% FBS and 10% DMSO.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003ePrimary Cell Line Establishment\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eFour fresh tumor fragments, tum_burn0214 (1.5 \u0026times; 1.5 mm each), were enzymatically digested with collagenase IV for 1 hour. After washing with phosphate-buffered saline (PBS), The resulting cell suspension was placed in a 6-well culture plate containing DMEM/F12 medium supplemented with 10% FBS, 2 mM Glutamax, and 1\u0026times; antibiotic/antimycotic, and maintained in a humidified atmosphere with 5% CO₂. The culture medium was replaced weekly. When the cell density reached 1\u0026ndash;1.5 million cells per milliliter (cells/mL), the cells were split to a concentration of 0.2\u0026ndash;0.3 million cells/mL and transferred to new culture flasks. Cryopreservation of the established cell line, lcl_burn0214, was performed at passages 6, 15, 35, 46, and 62 using DMEM/F12 supplemented with 45% FBS and 10% DMSO. Thawing tests were successfully conducted for all specified passages.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eTumor-Infiltrating Lymphocyte (TIL) TIL_burn0214 Expansion\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThawed tumor fragments, tum_burn0214 (5\u0026ndash;10 fragments per well of a 6-well plate), were placed in 2 mL of serum-free lymphocyte culture medium HIPP-T009 (Bioengine, Shanghai, China) supplemented with 5,000 units/mL IL-2 (Ronkoleukin, Biotech, Moscow, Russia) and 1\u0026times; antibiotic/antimycotic (TIL culture medium). The ImmunoCult\u0026trade; Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Vancouver, Canada) was added at a concentration of 25 \u0026micro;L/mL to activate T cells on day 0. Reactivation with the T Cell Activator was performed every 10\u0026ndash;12 days in accordance with the manufacturer\u0026rsquo;s protocol. The TIL medium was refreshed two days post-activation. Upon reaching a density of 1\u0026ndash;1.5 million cells/mL, TIL_burn0214 cells were subcultured into new flasks. Expanded TILs were subsequently characterized for phenotype and anti-EBV activity using the ELISPOT assay.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eIn vivo Tumorigenicity\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe tumorigenic potential of lcl_burn0214 cells was evaluated in vivo by subcutaneous injection into five immunodeficient male NU\u0026ndash;A/A Tyrc/Tyrc Foxn1nu/Foxn1nu mice (BALB/c nude strain, 6\u0026ndash;8 weeks old; obtained from the Laboratory Animals Breeding Facility BIBCh, Pushchino, Russia). Animals were maintained under specific pathogen-free conditions in the Vivarium of the Department of Experimental Biology at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (IBCh RAS). Animal procedures were approved by the local institutional review board of the Vivarium of the Department of Experimental Biology at the IBCh RAS under protocol #378/2023 and carried out in accordance with the institutional guidelines. Lcl_burn0214 cells at passage 14 were harvested and resuspended in phosphate-buffered saline at a concentration of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL. Each mouse received a subcutaneous injection of 0.1 mL cell suspension (total 1x10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells) into the right flank. Tumor growth was monitored weekly by measuring two orthogonal diameters with an electronic caliper. \u0026nbsp; Mice were euthanized 75 days post-injection. The only tumor that reached 200 mm\u0026sup3; was excised, fixed in 4% neutral-buffered formaldehyde, processed for paraffin embedding. Tissue sections (3.5 mkm) were prepared for hematoxylin and eosin staining.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eShort tandem repeats (STR) analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTIL_burn0214, PBMC_burn0214, the lcl_burn0214 cell line at passages 15, 53, from the patient were subjected to STR analysis. 27 loci were \u0026nbsp; amplified using PowerPlex\u0026reg; Fusion 6C System STR and detected by the Applied Biosystems 3500 Series Genetic Analyzer. Data were analyzed with GeneMapper ID‑X Software v1.6.\u003c/p\u003e\n\u003cp\u003eThe STR profile of the lcl_burn0214 cell line was compared with the Cellosaurus database to identify any related cell lines using the CLASTR 1.4.4 online tool [26].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003ePhenotypic analysis was performed on lcl_burn0214 and TIL_burn0214 cell cultures using the following monoclonal antibodies purchased from Sony Biotechnology, San Jose, Ca, USA: CD3_FITC (2186530), CD19_APC (2111055), and CD45_FITC (2120025), CD4_APC_cy7, CD8_PE (2105255), CD20_PE (2111525). DAPI was used to counterstain live cells. Cells (1x10\u003csup\u003e5\u003c/sup\u003e) were washed in PBS and resuspended in a staining buffer containing the appropriate antibodies. Surface marker staining was performed for 20\u0026thinsp;min at 4\u0026thinsp;\u0026deg;C. After incubation, cells were washed twice, collected in 100 \u0026mu;l of PBS, and immediately analyzed using the ACEA NovoCyte\u0026reg; flow cytometer VBR system (Agilent Technologies, Santa Clara, CA, USA). NovoCyte Express software was used for plot analysis.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eElispot assay\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo evaluate the functional response of tumor-infiltrating lymphocytes (TILs) to the EBV-positive autologous lcl_burn0214 cell line, co-culture experiments were performed. The TIL_burn0214 and lcl_burn0214 cell line were co-cultured at effector-to-target (E:T) ratios of 1:1 and 1:10 in duplicate wells of 96-well flat-bottom plates pre-coated with anti-IFN-\u0026gamma; antibodies. Each well contained either 5 000 or 50 000 TIL_burn0214 cells and 50 000 lcl_burn0214 cells. TIL_burn0214 cells were thawed and used immediately in the assay. Two independent vials of TIL_burn0214 were utilized as biological replicates.\u003c/p\u003e\n\u003cp\u003eAfter 18 hours of incubation, IFN\u0026gamma; production was quantified using a commercially available ELISpot kit (ImmunoSpot\u0026reg; ELISpot kit, Cellular Technology Limited, C.T.L., Cleveland, OH, USA), following the manufacturer\u0026rsquo;s protocol. Negative controls included wells containing either 50 000 lcl_burn0214 or 50 000 TIL_burn0214 cultured alone. Developed ELISpot plates were analyzed using the Immunospot analyzer software (C.T.L.), and the mean number of IFN-\u0026gamma; spot-forming units (SFUs) was calculated for each condition: TIL_burn0214 co-cultured with lcl_burn0214, TIL_burn0214 alone, and lcl_burn0214 alone.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eRNA extraction and construction of transcriptome libraries\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eRNA extraction from tumor tissue tum_burn0214 was performed using the All Prep RNA/DNA Mini Kit (Qiagen, Hilden, Germany). Tissue fragments preserved in Intact RNA solution were minced on a sterile Perti dish until they were as small as possible. Tissue fragments were then transferred to a 2 ml tube containing three stainless steel spheres of 4.8 mm diameter, to which 700 mcl of RLT lysis solution containing 1% beta-mercaptoethanol was added. Tissue fragments were homogenized using the Tissue Lyser instrument (Qiagen, Hilden, Germany) under the following conditions: 1 minute, 25/s. The next steps of RNA extraction were performed according to the manufacturer\u0026apos;s instructions for animal tissues.\u003c/p\u003e\n\u003cp\u003eRNA from lcl_burn0214 was also isolated using the All Prep RNA/DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026apos;s instructions for animal cells. Approximately 4*10\u003csup\u003e6\u003c/sup\u003e fresh pelleted cells were used as input. Homogenization was performed using a 20G needle with a syringe.\u003c/p\u003e\n\u003cp\u003eTotal RNA integrity number (RIN) was determined using RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA) with Bioanalyzer 2100 instrument (Agilent, USA). TURBO DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to remove DNA traces.\u003c/p\u003e\n\u003cp\u003eFor the construction of the transcriptome library, 500 ng of total RNA was collected. First, RNA was enriched for poly(A) fraction using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA). Next, non-directional transcriptome libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, USA) with the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (New England Biolabs, USA) according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003ePaired-end libraries were sequenced on the Illumina NovaSeq 6000 instrument at 2 \u0026times; 100 cycles (Illumina, San Diego, CA, USA).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eBioinformatics pipeline\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eData preparation for analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuality control by FastQC (0.12.1) [27] and MultiQC (1.15) [28] was performed before and after adapter trimming by cutadapt (version 4.4) [29] and quality filtering by trimmomatic (version 0.39) [30] for the whole data set. Pseudoalignment was performed by salmon (1.10.2) [31] for Gencode.v43 transcriptome with full genome decoy index. Summarizing for gene was performed by tximport (1.26.1) [32] using the length scaled TPM metrics and ignoring transcripts version. Expression and dispersion for genes and transcripts has been estimated by edgeR (version 3.40.2) [33]. For the further analysis CPM data from edgeR was used with filtering by maximal mean in any group \u0026gt; 4 CPM. PCA analysis was realized by PCAtools (v2.10.0) [34] on log2 transformed CPM removing genes with the lowest 10% variance. \u0026nbsp;Plots were plotted with ggplot2 (v3.4.3) [35].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eHLA Typing for RNA-seq data (tum_burn0214 and lcl_burn0214 samples)\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eHLA typing was performed with HLA-HD [36] against IMGT/HLA database (version 3.50) [37] using RNA-seq data from tum_burn0214 and lcl_burn0214 samples.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEBV reference genome aligning\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eRNA-seq reads were aligned to the human reference genome using STAR version 2.7.10b [38] in 2-pass mode. The reference genome used was the GRCh38 (hg38) assembly provided by the GDC [39], which includes decoy and 200 viral genomes including the EBV; the genome index for STAR was generated using GENCODE v43 gene annotations.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAnalysis of public cancer cell line transcriptomics data\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eLymphoma cell line transcriptomic data (Quentmeier H. dataset) \u0026nbsp;[40], were downloaded from ENA (PRJEB30312) [41], B95-8 EBV strain-infected lymphoblastoid B cell lines (Geuvadis consortium dataset) \u0026nbsp;[42] were downloaded from IGSR [43].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003ecell type deconvolution analysis\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eDeconvolution of cell types were realized by MSP, xCell and EPIC methods using the immunedeconv R package (v2.1.3) [44].\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eBCR profiling analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;BCR repertoire were evaluated by MiXCR (version 4.6.0) [45]\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch1\u003eData availability\u003c/h1\u003e\n\u003cp\u003eRNA-seq data produced in this study have been deposited at the BioProject and are available under accession number PRJNA1266483\u003c/p\u003e\n\u003ch1\u003eAcknowledgements\u003c/h1\u003e\n\u003cp\u003eThe authors thank core facilities of the Lopukhin FRCC PCM FMBA of Russia “Genomics, proteomics, metabolomics” () for performing sequencing\u003c/p\u003e\n\u003cp\u003eThe authors thank Julia A. Bespyatykh, the head of Laboratory of Molecular Medicine, Lopukhin FRCC PCM FMBA of Russia for EBV detection technical support\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors thank Marina S. Krasilshchikova the head of the Vivarium of the Department of Experimental Biology at the IBCh RAS for technical support with animal model experiments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors thank Anna Khalturina and LLC Impulstest for performing STR analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the \u0026nbsp;«T-kletki» \u0026nbsp; project funding 123032900030-7 by Federal Medical Biological Agency\u003c/p\u003e\n\u003ch1\u003eAuthor information\u003c/h1\u003e\n\u003cp\u003e\u003cu\u003eAuthors and Affiliations\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLopukhin FRCC PCM FMBA of Russia, Moscow, Russian Federation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTatiana V. Petrova, Daria V. Kuznetzova, Alexandra V. Kanygina, Liubov O. Skorodumova, Elena I. Sharova, Viktor A. Ivanov\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Burnasyan Federal Medical Biophysical Center FMBA of Russia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTatiana A. Astrelina, Svetlana E. Varlamova\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eContributions\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTVP EIS designed the study, TVP, DVK, LOS, VAI, EIS performed and interpreted experimental data, EIS, AVK, TVP performed and interpreted bioinformatic analysis, TAA, SEV provided tumor specimen and clinical data, TVP wrote the original draft with contributions from all authors. All authors edited the manuscript. The final version of the manuscript was approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCorresponding authors\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Tatiana V. Petrova [email protected], Elena I. Sharova [email protected]\u003c/p\u003e\n\u003ch1\u003eAdditional Information\u003c/h1\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003ch1\u003eEthics declarations\u003c/h1\u003e\n\u003cp\u003eFor tumor and blood samples from a patient this study was approved by the local institutional review board of the Burnasyan Federal Medical Biophysical Center FMBA of Russia under protocol RU-FMBC-23-07-2020. The study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from the patient prior to his participation.\u003c/p\u003e\n\u003cp\u003eAnimal procedures were approved by the local institutional review board of the Vivarium of the Department of Experimental Biology at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (IBCh RAS) under protocol #378/2023 and carried out in accordance with the institutional guidelines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSausen, D. G., Poirier, M. C., Spiers, L. M., Smith, E. N. Mechanisms of T cell evasion by Epstein-Barr virus and implications for tumor survival.\u003cem\u003e Frontiers in Immunology\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 1289313; \u003cu\u003e10.3389/fimmu.2023.1289313\u003c/u\u003e (2023).\u003c/li\u003e\n\u003cli\u003eLareau, C. A. et al. 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A. \u003cem\u003eet al.\u003c/em\u003e MiXCR: software for comprehensive adaptive immunity profiling. \u003cem\u003eNature Methods\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 380\u0026ndash;381 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6835907/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6835907/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecent studies underscore herpesvirus-associated risks, especially HHV6 and Epstein\u0026ndash;Barr virus (EBV), in CAR-T and tumor-infiltrating lymphocyte (TIL) therapies, partly due to ex vivo cell manipulation. EBV-driven B cell outgrowth can occur during TIL expansion, and EBV-transformed B cells may exert immunomodulatory functions, potentially impacting TIL therapy efficacy \u0026mdash; areas that remain underexplored. Here, we characterized a spontaneously arising wild-type EBV-transformed B cell line, lcl_burn0214, derived from rectal cancer tumor tissue during ex vivo culture. The lcl_burn0214 cell line has undergone 70 passages and was confirmed as a monoclonal B cell line of patient origin via HLA genotyping, immunophenotyping (CD19, CD20), B cell receptor clonotype analysis. RNA-seq analysis comparing EBV-positive and -negative B lymphoma and lymphoblastoid cell lines revealed that lcl_burn0214\u0026rsquo;s gene expression closely resembles that of lymphoblastoid cells rather than malignant B lymphomas. In immunocompromised mice, lcl_burn0214 exhibited limited tumorigenicity. The cell line expressed immune checkpoint genes (CD274, CSF1, TNFSF4). Nevertheless, autologous TILs were negative for B cells and exhibited specific anti-EBV activity, as demonstrated by the interferon-gamma response in the ELISPOT assay during coculture with lcl_burn0214. Our findings underscore the importance of monitoring TIL products for EBV-positive B cell contamination during manufacturing to ensure safety and therapeutic efficacy.\u003c/p\u003e","manuscriptTitle":"Spontaneous EBV-positive B cell line from rectal tumor: characterization, interaction with autologous tumor-infiltrating T lymphocytes, comparison with B lymphoma cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 05:42:37","doi":"10.21203/rs.3.rs-6835907/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-18T05:23:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T19:05:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T18:45:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308208496856225415194278049654907310496","date":"2025-06-27T12:33:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8775973650284470455343879735616461329","date":"2025-06-19T14:18:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T12:57:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-17T12:49:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-12T15:10:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-11T07:19:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-06T09:49:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c6228f12-92e2-43ae-b826-bbb228bf9663","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50257071,"name":"Biological sciences/Immunology/Immunotherapy"},{"id":50257072,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Rectal cancer"},{"id":50257073,"name":"Biological sciences/Microbiology/Virology/Herpes virus"}],"tags":[],"updatedAt":"2025-12-01T16:03:02+00:00","versionOfRecord":{"articleIdentity":"rs-6835907","link":"https://doi.org/10.1038/s41598-025-29456-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-24 15:58:03","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-06-23 05:42:37","video":"","vorDoi":"10.1038/s41598-025-29456-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-29456-7","workflowStages":[]},"version":"v1","identity":"rs-6835907","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6835907","identity":"rs-6835907","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}