High-risk EBV promotes immune evasion in nasopharyngeal carcinoma by upregulating HLA-DP via the encoded BALF2-HR variant

Domain: biology
Published: 2026-03-27
Editorial outcome: major_revision

Abstract
Nasopharyngeal carcinoma (NPC) is prevalent in East and Southeast Asia, with genetic factors playing a significant role in its occurrence. The HLA gene region on chromosome 6 is linked to NPC susceptibility, but the mechanisms remain unclear. Epstein-Barr virus (EBV) infection is a well-established cause, with 95% of NPC patients being EBV-positive. Three key variations in the EBV genome (162215_C, 162476_C, 163364_T) in the BALF2 gene are strongly associated with NPC risk. This study finds that the high-risk BALF2 variant (BALF2-HR) upregulates HLA class II molecules, such as HLA-DP, which interacts with LAG-3 on CD8⁺ T cells, inhibiting cytokine secretion and promoting T cell exhaustion, leading to immune evasion and reduced anti-PD-1 efficacy. BALF2-HR also enhances HLA-DP transcription by binding to KPNA2 and facilitating CIITA nuclear translocation. Conjunctive immunotherapy with anti-LAG-3 and anti-PD-1 antibodies significantly improves NPC treatment. This work introduces a new therapeutic strategy for NPC and insights into infection-associated cancers.

Introduction
EBV, the first recognized human oncogenic virus [1], infects over 90% of the world’s adult population. However, most infected individuals remain asymptomatic, with only a small fraction developing malignancies. The EBV infects human cells and expresses viral proteins and non-coding RNAs that promote cell proliferation, inhibit cell death, and enable immune evasion. Over time, these changes drive the malignant transformation of infected cells, ultimately leading to the formation of tumors [2]. EBV has been linked to various lymphomas, including Burkitt lymphoma [3], Hodgkin lymphoma [4], and NK/T-cell lymphoma [5], as well as epithelial malignancies such as NPC where over 95% of cases are EBV-positive and EBV-associated gastric carcinoma (EBVaGC), which constitutes 10% of gastric cancers [6], contributing to over 200,000 cases annually and 1.8% of cancer-related deaths worldwide. Among these EBV-associated cancers, only NPC displays a significant geographic concentration[7, 8]. NPC is most prevalent in North Africa, Southeast Asia, and southern China, accounting for approximately 50% of global NPC cases [6, 9, 10]. The etiology of NPC involves a combination of genetic predisposition, environmental and dietary carcinogens [2, 6, 11], which also suggests the presence of specific high-risk EBV subtype(s) that contribute to the elevated NPC rates in regions like southern China.

Early genomic analyses identified two major subtypes of EBV: Type 1, which is globally distributed, and Type 2, predominantly found in North Africa and possibly associated with EBV-induced lymphomas [12]. Utilizing next-generation sequencing, we uncovered a distinct EBV subtype prevalent among NPC patients in southern China [13, 14]. Xu et al. subsequently expanded the sample size and performed a case-control association study, confirming a high-risk EBV subtype strongly linked to NPC development [11]. This subtype is characterized by three specific mutations: 162215_C, 162476_C, and 163364_T, all located within the BamHI-A leftward reading frame 2 (BALF2) gene. This C-C-T high-risk subtype, termed BALF2-HR, is significantly associated with an increased risk of NPC [11]. BALF2 encodes a single-stranded DNA-binding protein that plays a role in the initiation of early lytic replication and viral particle formation [15-17], and is also expressed during EBV latency [17]. However, the exact mechanism by which the high-risk BALF2-HR variant contributes to NPC development, as opposed to the low-risk BALF2-LR variant, is unclear.

NPC tissues are often characterized by a substantial infiltration of lymphocytes, classifying them as "hot" tumors. Recently, significant progress has been made in NPC immunotherapy, particularly with immune checkpoint inhibitors such as monoclonal antibodies targeting PD-1/PD-L1, which have become the first-line treatment for recurrent or metastatic NPC [18]. However, around 70% of patients do not respond to anti-PD-1/PD-L1 monotherapy. These non-responders frequently exhibit brief response durations and develop resistance, suggesting that other immune checkpoint molecules may play crucial roles in immune evasion and resistance to immunotherapy in NPC [19]. Recent single-cell transcriptome analyses have identified a unique population of tumor cells within NPC tissues that exhibit dual "epithelial-immune" characteristics [20]. These NPC cells express classical epithelial markers, such as EpCAM, alongside immune molecules like HLA-II and complement proteins, which may enhance their ability to evade immune detection [20]. As early as 1990, Lu et al. established a strong linkage between the HLA locus and NPC susceptibility through family linkage analysis[21]. Bei et al. further identified several single-nucleotide polymorphisms (SNPs) within the HLA region associated with NPC risk through case-control genome-wide association studies (GWAS) [22]. However, the regulatory mechanism responsible for the high expression of HLA-II on NPC cells and the role of these molecules in promoting immune evasion and resistance to immunotherapy remains elusive.

In this study, we investigate the role of a high-risk EBV subtype, BALF2-HR, in the progression of nasopharyngeal carcinoma. We discover that BALF2-HR upregulates HLA-II expression, particularly HLA-DP, in NPC cells, which interacts with the immune checkpoint molecule LAG-3 on T cells, promoting T cell exhaustion and immune evasion. Our findings suggest that combining anti-LAG-3 and anti-PD-1 monoclonal antibodies significantly enhances the efficacy of NPC immunotherapy. These results not only improve our understanding of the molecular mechanisms behind NPC immune evasion but also propose a novel therapeutic approach that could optimize immunotherapy outcomes in NPC patients.

Results
1. BALF2-HR Enhances HLA-II Expression in NPC
Our previous whole-genome sequencing analysis of EBV from 26 NPC biopsy tissues from Hunan Province (a high-incidence region for NPC in southern China) [13, 14] revealed that 17 cases carried the C-C-T haplotype at the 162215, 162476, and 163364 loci, 3 cases had the C-C-C haplotype, 5 cases had the C-T-C haplotype, and 1 case had the A-C-C haplotype [Supplementary Fig.1A]. Through association analysis, Xu et al. identified the BALF2 C-C-T haplotype as the most significantly associated with NPC risk (designated as BALF2-HR), with an odds ratio of 11.71 compared to the low-risk BALF2 A-T-C variant (designated as BALF2-LR) (P = 2.39 × 10-24). The C-C-C haplotype showed a weaker association (odds ratio = 3.50, P = 1.22 × 10-5) [11].

In this study, we collected another 128 fresh NPC biopsy tissues from Hunan Province [Supplementary Data 1]. Using RT-PCR, we detected EBV in 110 of these samples, while the remaining 18 were EBV negative. Additionally, we collected paraffin-embedded tissue sections from 58 EBV-positive nasopharyngeal carcinoma patients [Supplementary Data 2] and identified the EBV BALF2 genotypes of these 168 NPC samples using Sanger sequencing, revealing that 124 cases had the C-C-T genotype, 17 cases were C-C-C, and 27 cases had other genotypes, further confirming that the C-C-T genotype is most strongly associated with NPC risk [Fig.1A]. In contrast, in NK/T-cell lymphoma, which also primarily affects the nasopharyngeal region but originates from a different cell type, 66.67% of the EBV BALF2 genotypes were low-risk A-T-C variant [23][Supplementary Fig.1B]. We performed immunohistochemical analysis on paraffin-embedded tissue sections from the aforementioned 58 cases of nasopharyngeal carcinoma and found that BALF2 is expressed in EBV-positive nasopharyngeal carcinoma tissues of both low-risk and high-risk subtypes. [Fig. 1B, Supplementary Fig.1C]. These results suggest that the BALF2 C-C-T genotype represents a relatively unique high-risk EBV subtype and is widely prevalent among nasopharyngeal carcinoma patients in southern China.

To investigate the biological function of EBV BALF2-HR in NPC pathogenesis, we constructed overexpression vectors for BALF2-HR and BALF2-LR (used as a control) and transfected them into the EBV-negative NPC cell line HK1 [Supplementary Fig.1D]. Differential gene expression analysis, conducted using RNA sequencing (RNA-seq) [Supplementary Data 3], revealed that BALF2-HR significantly upregulated the expression of HLA-II compared to BALF2-LR [Fig. 1C]. We further sequenced the BALF2 gene in the commonly used EBV-positive NPC cell line C666-1 and confirmed that it harbors the BALF2-HR (C-C-T) genotype [Supplementary Fig.1E]. Analysis of the whole genome expression profile (GSE194333) of C666-1 and immortalized EBV-negative nasopharyngeal epithelial cells NP69 also showed significantly higher expression of HLA-II in C666-1 cells [Fig. 1D]. Additionally, we analyzed single-cell sequencing data of 4 EBV-negative and 6 EBV-positive NPC biopsy tissues (GSE150825) [24], focusing on HLA-II expression. The results demonstrated that HLA-DPA1, DPB1, DQA1, DQB1, DRA, and DRB1 were significantly upregulated in EBV-positive NPC cells [Supplementary Fig.1F-H].

We also collected fresh human tissue samples from 25 cases of EBV-infected NPC [Supplementary Data 4]. A portion of these samples underwent Sanger sequencing to determine the genotype of the BALF2 gene, revealing that 16 cases carried the high-risk C-C-T subtype [Supplementary Fig.1I]. Another portion of the samples was dissociated into a single-cell suspension, and flow cytometry was used to assess the expression of HLA-II on the surface of NPC cells (EpCAM+). The results showed that HLA-II levels in epithelial cells from the 16 BALF2-HR NPC tissues were significantly higher than those from the 9 NPC tissues with other BALF2 subtypes [Fig. 1E].

In addition to the high-risk EBV-positive NPC cell line C666-1, we also obtained two other cell lines, HONE1-EBV and HK1-EBV, which were infected with the recombinant Akata EBV, kindly provided by Prof. Sai Wah Tsao of the University of Hong Kong [25]. Sanger sequencing revealed that the Akata EBV carries the low-risk A-T-C BALF2 subtype [Supplementary Fig.1J]. RT-PCR, Western blot, and immunofluorescence assays confirmed the expression of BALF2 mRNA [Supplementary Fig.1K] and protein [Supplementary Fig.1L-M] in all three cell lines. Subsequent Western blot [Supplementary Fig.2A] and immunofluorescence experiments [Supplementary Fig.2B] showed that the expression of HLA-II molecules (HLA-DP, HLA-DQ, and HLA-DR) was significantly higher in C666-1 cells carrying BALF2-HR compared to EBV-negative (HONE1, HK1) or BALF2-LR expressing (HONE1-EBV, HK1-EBV) NPC cell lines. Of note, knockout of BALF2 using sgRNA in C666-1 cells carrying high-risk EBV and in HK1-EBV and HONE1-EBV cells infected with low-risk EBV did not alter the latent state of EBV [Supplementary Fig.2C]. However, overexpression of BALF2-HR or BALF2-LR in the EBV-negative NPC cell lines HK1 and HONE1 [Supplementary Fig.2D], or knocking out BALF2 in C666-1, HK1-EBV, and HONE1-EBV cells, showed that only overexpressing or knocking out BALF2-HR can significantly upregulate or downregulate the expression of classical HLA-II in NPC cells, as revealed by the results of RT-qPCR [Fig. 1F, Supplementary Fig.2E], Western blot [Fig. 1G, Supplementary Fig.2F], immunofluorescence [Fig. 1H, Supplementary Fig.2G], and flow cytometry [Fig. 1I, Supplementary Fig.2H]. In contrast, knocking out BALF2-LR in HONE1-EBV and HK1-EBV did not result in a significant change in HLA-II [Supplementary Fig.3A-C]. These results suggest that the high-risk EBV upregulates HLA-II expression in NPC cells via BALF2-HR, whereas BALF2-LR has no significant effect on HLA-II expression.

2. BALF2-HR Drives Immune Evasion in NPC and Contributes to Anti-PD-1 Immunotherapy Resistance
HLA class II molecules are primarily expressed on the surface of antigen-presenting cells, where their peptide-binding groove (comprising the α₁ and β₁ domains) binds exogenous antigenic peptides. CD4⁺ T cells recognize the HLA class II-peptide complex through their T-cell receptor (TCR), thereby initiating an immune response [26]. However, recent studies have shown that they can also bind to the inhibitory immune checkpoint molecule LAG-3 on T cells, thereby suppressing T cell function [27-30]. Using flow cytometry, we analyzed the aforementioned 16 NPC biopsy samples infected with high-risk EBV and 9 samples with non-high-risk EBV [Supplementary Data 3]. In NPC tissues infected with high-risk EBV, we observed a significantly reduced proportion of CD8+ T cells, along with decreased expression of cytotoxic cytokines in these cells, indicative of a pronounced exhausted phenotype. Notably, the proportions of CD4+ T cells, regulatory T cells (Tregs), Th1 cells, Th2 cells, Th17 cells, M2 macrophages, B cells, and NK cells were not significantly altered. These findings suggest that high-risk EBV subtypes may selectively impair CD8+ T cell function and promote their exhaustion [Fig. 2A, Supplementary Fig.4A-D].

To further assess the impact of HLA class II molecules on tumor cells in modulating CD4⁺ T cell responses, human PBMCs were isolated, and primary CD4⁺ T cells were purified using CD4 microbeads. These CD4⁺ T cells were co-cultured for a week with dendritic cells and irradiated NPC cells to generate NPC-reactive CD4⁺ T cells [31], and were later HLA-DR matched with nasopharyngeal carcinoma cells to prevent immune rejection. Subsequently, BALF2-HR or BALF2-LR was overexpressed in HK1 and HONE1 cells, while BALF2-HR was knocked out in C666-1 cells. These modified tumor cells were then co-cultured with NPC-reactive CD4⁺ T cells. Flow cytometry analysis of IL-2, Ki-67, CD69, and FOXP3 expression in CD4⁺ T cells revealed that the high expression of tumor cell HLA-II molecules, mediated by BALF2-HR, partially promoted CD4⁺ T cell activation [Supplementary Fig.4E], as previously reported [32].

To clarify the interaction between HLA class II molecules and CD8⁺ T cells, primary CD8⁺ T cells were isolated from human PBMCs using CD8 microbeads. These cells were then co-cultured with dendritic cells and irradiated NPC cells for one week to produce NPC-reactive CD8⁺ T cells. Subsequently, NPC-reactive CD4⁺ T cells and NPC-reactive CD8⁺ T cells were co-cultured with tumor cells expressing BALF2-HR. Tumor cell viability, assessed by crystal violet staining, indicated that BALF2-HR exerts its effect predominantly through CD8⁺ T cells rather than CD4⁺ T cells [Supplementary Fig.4F]. This conclusion was further supported by flow cytometry analysis of tumor cell apoptosis [Supplementary Fig.4G]. Single-cell RNA sequencing data from the GSE150430 dataset showed that LAG-3 is mainly expressed on CD8⁺ T cells, with relatively lower expression on CD4⁺ T cells [Supplementary Fig.4H]. These results indicate that in the absence of CD8⁺ T cells, BALF2-HR-mediated CD4⁺ T cell activation is unable to suppress the growth of nasopharyngeal carcinoma cells. Furthermore, flow cytometry and co-culture experiments on nasopharyngeal carcinoma tissues both indicate that BALF2-HR suppresses the cytotoxic function of CD8⁺ T cells. Future studies will investigate the interaction between BALF2-HR and CD8⁺ T cells.

The 58 NPC tissue paraffin sections used in Fig. 1B were from patients who received a combination of conventional treatment and anti-PD-1 (Nivolumab) therapy [Supplementary Data 2]. Of these, 39 cases harbored the high-risk C-C-T BALF2, while the remaining 19 cases had non-high-risk BALF2 [Supplementary Fig.5A]. Further analysis of the relationship between BALF2 genotypes and immunotherapy efficacy revealed that among the 39 patients with the BALF2-HR subtype, only 9 achieved a complete response (CR), resulting in a CR rate of 23.08%. Additionally, 12 patients showed a partial response (PR), yielding a PR rate of 30.77%. In contrast, among the 19 patients with non-high-risk EBV subtypes, 8 achieved CR (42.10%), and 9 had PR (47.37%). The overall objective response rate (CR + PR) was 53.85% in the BALF2-HR group compared to 89.47% in the non-high-risk group (P = 0.0043) [Fig. 2B]. Immunohistochemical analysis of BALF2 expression in these tissues revealed that only the high-risk BALF2 expression level showed a significant positive correlation with the efficacy of anti-PD-1 therapy. The mean expression value for PD + SD was 8.3, while the mean expression value for PR + CR was 4.8 [Fig. 2B]. These results suggest that the BALF2-HR promotes immune evasion in NPC by upregulating HLA-II and inducing CD8+ T cell exhaustion, which may contribute to resistance to anti-PD-1 immunotherapy.

To further validate our hypothesis, we overexpressed BALF2-LR or BALF2-HR in EBV-negative NPC cell lines HONE1 and HK1 or knocked out BALF2 in BALF2-HR-infected NPC cell line C666-1 using sgRNA. We found that neither overexpression nor knockout of BALF2-HR affected the proliferation of NPC cells [Supplementary Fig.5B]. Next, we isolated CD8+ T cells and co-cultured them with the aforementioned NPC cells [Supplementary Fig.5C]. The results showed that overexpression of BALF2-HR significantly enhanced NPC cell survival in co-culture with CD8+ T cells, while knocking out BALF2-HR significantly reduced NPC cell survival. When we added a PD-1 monoclonal antibody (nivolumab) to the culture medium, along with an IgG control, there was no significant change in BALF2-HR-induced NPC cell survival. However, combining PD-1 and LAG-3 monoclonal antibodies (Relatlimab) significantly reduced BALF2-HR-induced NPC cell survival [Fig. 2C, Supplementary Fig.5D]. We also used flow cytometry to assess the apoptosis of NPC cells from the same experiments. Overexpression of BALF2-HR significantly inhibited CD8+ T cell-induced apoptosis of NPC cells, while knockout of BALF2-HR significantly promoted NPC cell apoptosis. Adding the PD-1 monoclonal antibody alone did not affect the reduction in apoptosis mediated by BALF2-HR, but the combined use of PD-1 and LAG-3 monoclonal antibodies significantly reversed the reduction in NPC cell apoptosis mediated by BALF2-HR [Fig. 2E, Supplementary Fig.5E].

In the co-culture model of NPC cells and CD8+ T cells, we also assessed T cell functionality and activity using flow cytometry. NPC cells expressing BALF2-HR significantly reduced the proportion of CD8+ T cells expressing effector cytokines IFN-γ+ GZMB+ and IFN-γ+ TNF+, while knocking out BALF2-HR restored the proportion of these cytokine-expressing CD8+ T cells. The addition of PD-1 monoclonal antibody alone did not reverse BALF2-HR-mediated reduction in cytokine expression in CD8+ T cells, but the combined use of PD-1 and LAG-3 monoclonal antibodies effectively restored cytokine expression [Fig. 2F, Supplementary Fig.5F]. ELISA [Fig. 2G, Supplementary Fig.5G] and RT-qPCR [Supplementary Fig.5H] assays measuring the expression of effector cytokines IFN-γ, GZMB, and TNF in CD8+ T cells yielded similar results.

During the exhaustion of CD8+ T cells, not only is there a downregulation of effector cytokine expression, but also an increase in the expression of inhibitory receptors, such as PD-1 and TIM-3 [31, 32]. Flow cytometry confirmed that the proportion of PD-1+TIM-3+ cells among CD8+ T cells increased after co-culture with BALF2-HR-expressing NPC cells, while this proportion decreased when BALF2-HR was knocked out. Adding PD-1 monoclonal antibody alone did not reverse BALF2-HR-mediated CD8+ T cell exhaustion, but the combined use of PD-1 and LAG-3 monoclonal antibodies significantly inhibited the exhaustion of CD8+ T cells [Fig. 2H, Supplementary Fig.5I]. RT-qPCR [Supplementary Fig.5J] examination of the expression of CD8+ T cell exhaustion-related genes PDCD1, TIM-3, and TOX also supported these findings. These results suggest that BALF2-HR promotes immune evasion and resistance to immunotherapy in NPC by upregulating HLA class II molecules on NPC cells, leading to CD8+ T cell exhaustion and impaired cytotoxic function. LAG-3 monoclonal antibody restores T cell function, counteracts CD8+ T cell exhaustion, and enhances the efficacy of PD-1 monoclonal antibody therapy.

3. BALF2-HR Induces Immune Evasion and Immunotherapy Resistance in NPC via Upregulation of HLA-DP
Class II HLA molecules, including HLA-DP, HLA-DQ, and HLA-DR, are crucial components in immune regulation. To further investigate their roles in immune evasion and immunotherapy resistance in NPC, we overexpressed or knocked out HLA-DP, HLA-DQ, and HLA-DR in NPC cell lines [Supplementary Fig.6A-B]. Crystal violet staining, used to assess tumor cell survival, showed that these HLA-II molecules do not directly influence tumor cell proliferation [Supplementary Fig.6C]. However, in co-culture with CD8+ T cells, overexpression of HLA-DP in NPC cells significantly increased resistance to CD8+ T cell-mediated cytotoxicity. In contrast, overexpression of HLA-DQ or HLA-DR had no impact on the survival of co-cultured NPC cells. Using sgRNA to knock out these HLA-II molecules, we found that knocking out HLA-DP significantly reduced the survival of co-cultured NPC cells, while the knockout of HLA-DQ or HLA-DR did not notably affect NPC cell survival. Furthermore, adding PD-1 monoclonal antibody alone to the co-culture system did not affect HLA-DP-mediated survival of NPC cells. However, the combined use of PD-1 and LAG-3 monoclonal antibodies significantly inhibited the survival of HLA-DP-expressing NPC cells [Fig. 3A, Supplementary Fig.6D]. Similarly, Annexin V-PI staining revealed that overexpression of HLA-DP decreased NPC cell apoptosis, while its knockout increased apoptosis in the co-culture system [Fig. 3B, Supplementary Fig.6E]. These results indicate that, among classical HLA-II molecules, HLA-DP plays a key role in promoting immune evasion in NPC and contributes to resistance against PD-1 therapy. Since LAG-3 serves as a receptor for HLA-II molecules, its monoclonal antibody can inhibit HLA-DP-mediated immune evasion in NPC cells, thereby enhancing the efficacy of PD-1 immunotherapy.

Co-culturing NPC cells with CD8+ T cells and analyzing via flow cytometry [Fig. 3C, Supplementary Fig.6F] demonstrated that overexpression of HLA-DP in NPC cells significantly reduced the proportion of CD8+ T cells expressing the effector cytokines IFN-γ+ GZMB+ and IFN-γ+ TNF+. Knocking out HLA-DP expression in NPC cells using sgRNA restored the proportions of these cytokine-expressing CD8+ T cells. The addition of PD-1 monoclonal antibody alone did not reverse the HLA-DP-mediated reduction in CD8+ T cell effector cytokine expression. However, the combined use of PD-1 and LAG-3 monoclonal antibodies effectively restored effector cytokine expression in CD8+ T cells. ELISA [Supplementary Fig.6G-H] and RT-qPCR [Supplementary Fig.6I-J] assays for IFN-γ, GZMB, and TNF in CD8+ T cells support these results. Further flow cytometry analysis [Fig. 3D, Supplementary Fig.6K] showed that NPC cells overexpressing HLA-DP increased the proportion of PD-1+TIM-3+ cells among CD8+ T cells, indicative of T cell exhaustion, while knocking out HLA-DP reduced the proportion of these exhausted CD8+ T cells. The addition of PD-1 monoclonal antibody alone did not reverse HLA-DP-mediated CD8+ T cell exhaustion, but the combined use of PD-1 and LAG-3 monoclonal antibodies successfully reversed this effect. RT-qPCR [Supplementary Fig.6L-M] for T cell exhaustion markers such as PDCD1, TIM-3, and TOX, also yielded consistent findings. These results further confirm that HLA-DP plays a pivotal role in immune evasion in NPC by inhibiting T cell cytotoxic function and promoting T cell exhaustion. The combined use of PD-1 and LAG-3 monoclonal antibodies can counteract the inhibitory effects of HLA-DP on T cells, offering a promising strategy to improve immunotherapy efficacy.

In the above study, we used the NCBI reference sequences for HLA-DP, specifically HLA-DPA101:03 and HLA-DPB104:01. Given the population-specific distribution of HLA-DP alleles, deep sequencing of the MHC-II region in the Chinese population [33] revealed that the predominant HLA-DPA1 alleles are *02:02 (48.54%), *01:03 (39.39%), and *02:01 (11.35%), while the major HLA-DPB1 alleles are *05:01 (36.86%), *02:01 (19.74%), and *04:01 (10.60%). To investigate the functional diversity among HLA-DP alleles, we synthesized plasmids encoding the relevant variants and overexpressed them in the HK1 and C666-1 nasopharyngeal carcinoma (NPC) cell lines. These alleles did not significantly affect tumor cell proliferation [Supplementary Fig.7A-B]. However, co-culture of HLA-DP–overexpressing tumor cells with CD8+ T cells revealed that different HLA-DP alleles enhanced tumor cell survival, as shown by crystal violet staining [Supplementary Fig.7C] and supported by flow cytometry analysis of tumor cell apoptosis [Supplementary Fig.7D]. Further flow cytometry analysis of CD8⁺ T cells revealed that various HLA-DP alleles differentially suppressed cytotoxic cytokine production and promoted T cell exhaustion [Supplementary Fig.7E-F]. Together, these results suggest that HLA-DP alleles contribute to immune evasion in NPC to varying degrees, potentially through allele-specific amino acid variations that alter the structure of the antigen-binding groove and affect its interaction with LAG-3.

We overexpressed BALF2-HR in the EBV-negative NPC cell lines HONE1 and HK1 while knocking out HLA-DP, HLA-DQ, or HLA-DR individually. Additionally, in the high-risk EBV-infected NPC cell line C666-1, we knocked out BALF2-HR and overexpressed HLA-DP, HLA-DQ, or HLA-DR. Proliferation of these NPC cells remained unaffected [Supplementary Fig.8A]. When co-cultured with CD8+ T cells and assessed by crystal violet staining, we found that knocking out HLA-DP expression significantly inhibited BALF2-HR-mediated NPC cell survival. Conversely, overexpression of HLA-DP reversed the decreased tumor cell survival caused by BALF2-HR knockout. In NPC cells overexpressing BALF2-HR and simultaneously knocking out HLA-DP, the addition of PD-1 monoclonal antibody enhanced T cell-mediated cytotoxicity against NPC cells. However, overexpression of HLA-DP reversed the increased sensitivity of BALF2-HR knockout tumor cells to PD-1 monoclonal antibody treatment, leading to increased NPC cell survival and resistance to anti-PD-1 immunotherapy. The combined use of PD-1 and LAG-3 monoclonal antibodies further enhanced T cell-mediated killing of NPC cells [Fig. 4A-B, Supplementary Fig.8B]. Flow cytometry analysis of NPC cell apoptosis under these various treatment conditions yielded similar results [Fig. 4C, Supplementary Fig.8C].

Flow cytometry analysis of T cell function and activity [Fig. 4D, Supplementary Fig.8D] demonstrated that overexpression of BALF2-HR in NPC cells decreased the cytotoxic function of co-cultured CD8+ T cells. However, knocking out HLA-DP in BALF2-HR-overexpressing NPC cells reversed this suppression of T cell cytotoxic function. Overexpression of HLA-DP in C666-1 cells with BALF2-HR knockout reversed the enhanced CD8+ T cell cytotoxic function observed with BALF2-HR knockout. Additionally, NPC cells lacking HLA-DP exhibited increased sensitivity to PD-1 monoclonal antibody treatment, while cells overexpressing HLA-DP became resistant to PD-1 blockade. The combination of PD-1 and LAG-3 monoclonal antibodies resulted in significantly improved T cell-mediated killing of NPC cells compared to PD-1 treatment alone. ELISA [Fig. 4E, Supplementary Fig.8E] and RT-qPCR [Supplementary Fig.8F] analyses of CD8+ T cell cytotoxic cytokines, including IFN-γ, GZMB, and TNF produced similar findings. Further flow cytometry analysis [Fig. 4F, Supplementary Fig.8G] confirmed that either knocking out HLA-DP or adding LAG-3 monoclonal antibody could reverse BALF2-HR-mediated CD8+ T cell exhaustion and resistance to PD-1 therapy. RT-qPCR [Supplementary Fig.8H] showed comparable results for exhaustion markers PDCD1, TOX, and TIM-3 in CD8+ T cells. These findings indicate that HLA-DP plays a crucial role in mediating BALF2-HR-induced immune evasion in NPC.

HLA-II molecules not only serve as classical ligands for LAG-3 but also act as co-ligands for CD4 [30]. Due to the structural similarity between LAG-3 and CD4, it is possible that CD4 could competitively bind to both LAG-3 and HLA-II. To explore whether CD4 competes with LAG-3 for HLA-II binding, we first overexpressed or knocked out HLA-DP, HLA-DQ, and HLA-DR in NPC cells and co-cultured them with NPC-reactive CD4+ T cells. Flow cytometry analysis revealed that HLA-II expression on tumor cells had a modest inhibitory effect on CD4⁺ T cell differentiation, proliferation, and activation [Supplementary Fig.9A]. This suggests that HLA-II preferentially binds to LAG-3 on CD8+ T cells rather than to CD4 on CD4+ T cells, potentially due to differences in binding affinities. To further assess the binding affinity of HLA-II to LAG-3 and CD4, we overexpressed HLA-II in NPC cells and added 1 µg/mL of recombinant LAG-3 and CD4 proteins to the culture medium [Supplementary Fig.9B]. Immunofluorescence [Supplementary Fig.9C] and flow cytometry [Supplementary Fig.9D] confirmed that HLA-II on the surface of NPC cells predominantly binds LAG-3. These findings suggest that HLA-II in NPC cells preferentially interacts with LAG-3 on CD8+T cells rather than CD4.

To assess the binding affinity of HLA-DP, HLA-DQ, and HLA-DR to LAG-3 in NPC cells, recombinant proteins of each HLA class II molecule were individually co-incubated with LAG-3. Bio-layer interferometry (BLI) binding assays revealed that HLA-DP binds to LAG-3 with significantly higher affinity than HLA-DQ and HLA-DR [Fig. 5A, Supplementary Fig.10A]. We individually overexpressed these HLA-II molecules in NPC cells and added 1 µg/mL of recombinant LAG-3 protein to the medium, simulating in vitro interactions between HLA-DP, HLA-DQ, HLA-DR, and LAG-3 [Supplementary Fig.10B]. Results of immunofluorescence [Fig. 5B, Supplementary Fig.10C-D] and flow cytometry [Supplementary Fig.10E-F] showed that NPC cells expressing HLA-DP bound significantly more recombinant LAG-3 protein compared to cells overexpressing HLA-DQ or HLA-DR. Furthermore, when we simultaneously overexpressed HLA-DP, HLA-DQ, and HLA-DR in NPC cell lines, added 1 µg/mL recombinant LAG-3 protein, and incubated for 6 hours, co-immunoprecipitation (Co-IP) assays using HLA-DP, HLA-DQ, HLA-DR, or LAG-3 antibodies confirmed the immunofluorescence and flow cytometry results [Supplementary Fig.10G-H]. Among the HLA-II molecules tested, HLA-DP exhibited the strongest affinity for LAG-3, suggesting that NPC-produced HLA-DP may facilitate T cell exhaustion by binding to LAG-3 on CD8+ T cells.

To further elucidate the molecular mechanism by which LAG-3 inhibits T cell function, we focused on its activation via ubiquitination, a modification known to be essential for LAG-3's immunosuppressive activity [34]. BALF2-HR was overexpressed in HONE1 and HK1 tumor cells or knocked out in C666-1 cells, which were then co-cultured with CD8⁺ T cells. In parallel, either IgG or a LAG-3 monoclonal antibody (mAb) was added to the cultures. Co-immunoprecipitation (Co-IP) assays revealed that overexpression of BALF2-HR significantly enhanced LAG-3 ubiquitination in CD8⁺ T cells, whereas knockout of BALF2-HR markedly reduced it. Notably, the LAG-3 mAb blocked the BALF2-HR–induced increase in LAG-3 ubiquitination [Fig. 5C]. Lck (lymphocyte-specific protein tyrosine kinase) is a critical component of the T cell receptor (TCR) signaling pathway. Previous studies have shown that LAG-3 interferes with the interaction between CD8 and Lck [35, 36]. Co-IP assays demonstrated that BALF2-HR overexpression in tumor cells inhibited CD8–Lck binding, thereby attenuating downstream TCR signaling. This effect was reversed by treatment with LAG-3 mAb [Fig. 5D]. These findings suggest that BALF2-HR interferes with the interaction between CD8 and Lck by promoting the ubiquitination of LAG-3, potentially leading to additional inhibition of downstream TCR signaling.

Phosphorylation of ZAP70 functions as a critical "molecular switch" in TCR signal transduction, while LAG-3 has been shown to promote the expression of the exhaustion-associated transcription factor TOX [37]. Western blot analysis of TCR signaling components revealed that BALF2-HR overexpression reduced ZAP70 phosphorylation and upregulated TOX expression. Notably, treatment with a LAG-3 mAb restored ZAP70 phosphorylation and suppressed TOX levels [Fig. 5E]. Collectively, these findings demonstrate that BALF2-HR promotes LAG-3 activation via ubiquitination, thereby disrupting the CD8–Lck interaction and attenuating TCR signaling by decreasing ZAP70 phosphorylation. This suppression of TCR signaling lifts the inhibition of TOX, ultimately driving CD8⁺ T cell exhaustion. This mechanistic insight supports the rationale for combination immunotherapy targeting both LAG-3 and PD-1.

To examine how tumor cell HLA-II molecules regulate CD4⁺ T cell TCR signaling, we co-cultured CD4⁺ T cells with tumor cells. We observed that tumor cell HLA-II molecules could enhance the activation of CD4⁺ T cell TCR signaling [Supplementary Fig.10I]. Furthermore, these results suggest that MHC-II-LAG3 interactions are not a primary mechanism of CD4+ T cell suppression, even though anti-LAG3 mAb increased ZAP70 signaling. Therefore, other suppressive pathways involving LAG3 may also affect CD4+ T cells. This suggests that, in CD4⁺ T cells, LAG-3 expression is also a crucial factor restricting TCR pathway activation, which warrants further investigation.

We conducted immunohistochemical analysis on paraffin-embedded tissue samples from 58 NPC patients who received conventional treatment combined with PD-1 immunotherapy, as shown in Fig. 1B [Supplementary Data 2], to assess the relationship between BALF2 and HLA-DP expression levels. Our findings indicate a significant positive correlation between BALF2-HR expression and HLA-DP expression in NPC patients [Fig. 5F]. Among these 58 patients, 37 exhibited effective treatment outcomes (PR + CR), while 21 did not respond to treatment (PD + SD). Further analysis revealed that HLA-DP expression was significantly higher in patients who did not respond to treatment compared to those who had effective treatment outcomes [Fig. 5G].

4. BALF2-HR Interaction with KLPNA2 Facilitates CIITA Nuclear Translocation and Upregulation of HLA-DP
To investigate how BALF2-HR upregulates HLA-DP expression, we overexpressed either BALF2-HR or BALF2-LR in the EBV-negative NPC cell line HK1. Co-IP [Fig. 6A] assays combined with high-throughput mass spectrometry identified 318 proteins binding to BALF2-HR and 314 proteins binding to BALF2-LR, with 68 proteins uniquely interacting with BALF2-HR [Supplementary Data 5]. Analysis of gene expression profiles for these 68 proteins in the NPC clinical sample dataset GSE12452 and their correlation with HLA-DP expression revealed that KPNA2, TUBA4B, and MRPL30 were strongly correlated with HLA-DP expression [Supplementary Fig.11A]. Subsequent Co-IP experiments confirmed that BALF2-HR has a strong binding affinity for KPNA2 [Supplementary Fig.11B-C]. Protein-protein docking analysis using Alphafold2 [38] and MOE [39] [Supplementary Fig.11D] predicted that KPNA2 has a higher affinity for BALF2-HR than for BALF2-LR. Using truncation mutants based on Alphafold2-predicted binding sites, Co-IP experiments [Fig. 6B, Supplementary Fig.11E] demonstrated that BALF2-HR binds the full-length KPNA2 but not its truncation mutants. Further transfection experiments with BALF2-HR and its truncation mutants into HONE1 and HK1 cells confirmed [Fig. 6C] that the 517-850aa (MT-2) region of BALF2-HR is critical for binding to KPNA2, aligning with the binding site prediction from Alphafold2.

KPNA2, a member of the nuclear transport protein family, plays a critical role in the nuclear translocation of various transcription factors and is crucial for tumor formation and progression [40]. The class II major histocompatibility complex transactivator (CIITA) is a key regulator of HLA-II expression [41]. To investigate whether BALF2-HR upregulates HLA-II by binding to KPNA2 and facilitating the nuclear translocation of CIITA, we conducted protein-protein docking analyses using Alphafold2 and MOE. These analyses indicated a low affinity between KPNA2 and CIITA alone, but the affinity significantly increased when KPNA2 was complexed with BALF2-HR [Supplementary Fig.11F]. Co-IP experiments confirmed that BALF2-LR did not bind to KPNA2 or CIITA in HONE1-EBV and HK1-EBV cells, while BALF2-HR bound to both KPNA2 and CIITA in C666-1 cells [Supplementary Fig.11G]. Overexpression of BALF2-HR or BALF2-LR in EBV-negative NPC cells HONE-1 and HK1 further validated that BALF2-HR binds to both KPNA2 and CIITA, but BALF2-LR does not. Additionally, without BALF2-HR, KPNA2 and CIITA did not interact [Fig. 6D].

Nuclear-cytoplasmic fractionation experiments revealed that BALF2-LR, KPNA2, and CIITA were primarily located in the cytoplasm in NPC cells overexpressing BALF2-LR. However, in cells overexpressing BALF2-HR, these proteins were predominantly found in the nucleus. Knockdown of BALF2-HR in C666-1 cells, which are infected with a high-risk EBV subtype, significantly reduced the nuclear presence of both KPNA2 and CIITA [Fig. 6E, Supplementary Fig.11H]. Immunofluorescence assays confirmed these findings [Supplementary Fig.11I-J]. Furthermore, knockdown of KPNA2 in BALF2-HR-overexpressing NPC cells resulted in a decreased nuclear distribution of BALF2-HR and CIITA. Similar results were observed in C666-1 cells following KPNA2 knockdown [Fig.6F, Supplementary Fig.11K]. These results suggest that BALF2-HR facilitates CIITA nuclear translocation by binding to KPNA2.

Additionally, Western blot [Fig.6G, Supplementary Fig.11L], flow cytometry [Fig.6H, Supplementary Fig.11M-N], and RT-qPCR [Supplementary Fig.11O] analyses showed that KPNA2 knockdown significantly reversed the upregulation of HLA-DP, HLA-DQ, and HLA-DR induced by BALF2-HR. These findings identify KPNA2 as a key downstream mediator in BALF2-HR-driven upregulation of HLA-II, indicating that BALF2-HR promotes the transcription of HLA class II molecules by binding to KPNA2 and facilitating the nuclear translocation of CIITA.

5. Combination of LAG-3 and PD-1 Antibodies Enhances Immunotherapy Efficacy in NPC
We demonstrated that BALF2-HR promotes immune evasion and resistance to immunotherapy in NPC cells by upregulating HLA-DP, which binds to LAG-3 on T cells. To assess whether anti-LAG-3 can enhance the efficacy of anti-PD-1 treatment in NPC, we overexpressed BALF2-LR, BALF2-HR, or an empty vector in the EBV-negative NPC cell line HK1 and knocked out BALF2-HR in the EBV high-risk NPC cell line C666-1 using sgRNA, with scrambled sgRNA as a negative control. These five groups of differently treated NPC cells were subcutaneously injected into the right hind limbs of nude mice, which lack functional T cells, to establish xenograft models (40 mice per group). Seven days after injection, each group of tumor-bearing mice was divided into four subgroups (10 mice per subgroup). One subgroup received tail vein injections of PBS, while the other three subgroups received tail vein injections of T cells (5×107 cells/mouse) stained with the live-cell fluorescent dye DIR for tracking. The T cells were prepared through co-culture with dendritic cells stimulated by tumor cell lysates to simulate antigen presentation [42]. The three T-cell-receiving subgroups were treated with IgG (50 µg/mouse, negative control), anti-PD-1 (50 µg/mouse), or a combination of anti-PD-1 (50 µg/mouse) and anti-LAG-3 (50 µg/mouse), simulating immunotherapy [Supplementary Fig.12A].

Live animal imaging seven days after T cell infusion showed minimal T cell distribution within the xenograft tumors overexpressing BALF2-HR. Treatment with anti-PD-1 alone did not restore T cell infiltration in these tumors. However, the combination of anti-PD-1 and anti-LAG-3 significantly increased T cell accumulation at the xenograft site. Similarly, BALF2-HR knockout in C666-1 xenografts led to a notable increase in T cell infiltration. Both anti-PD-1 monotherapy and the combination of anti-PD-1 and anti-LAG-3 further enhanced T cell distribution in these xenografts. These findings suggest that BALF2-HR inhibits T cell survival in tumor tissues, whereas the combined use of LAG-3 and PD-1 antibodies enhances T cell survival in NPC tissues that overexpress BALF2-HR [Fig. 7A-B].

Furthermore, tumor growth was monitored in real-time, and mice were euthanized 5-6 weeks later to collect the tumor tissues [Fig. 7C, Supplementary Fig.12B-C]. No significant differences in tumor volume or weight were observed between the PBS-treated groups overexpressing BALF2-LR, BALF2-HR, or BALF2-HR knockout. However, in the T cell-treated groups, BALF2-HR overexpression led to larger xenograft tumors compared to controls, while BALF2-HR knockout resulted in significantly smaller tumors. BALF2-HR overexpression conferred resistance to anti-PD-1 therapy, resulting in larger tumor volumes, whereas BALF2-HR knockout increased sensitivity to anti-PD-1, reducing tumor size. The combination of anti-PD-1 and anti-LAG-3 provided the most effective tumor suppression. Flow cytometry of single-cell suspensions from selected xenograft tissues (6 mice per group) in the T cell-treated groups revealed that BALF2-HR overexpression decreased CD8+ T cell numbers and increased their exhaustion. Conversely, BALF2-HR knockout increased CD8+ T cell numbers and reduced exhaustion. Anti-PD-1 treatment alone did not reverse BALF2-HR-mediated CD8+ T cell depletion and exhaustion, but the combination of anti-PD-1 and anti-LAG-3 did [Fig. 7D]. ELISA [Supplementary Fig.12D] and RT-qPCR [Supplementary Fig.12E-F] results further supported these findings.

Paraffin-embedded xenograft tissues from the groups described above were analyzed by ISH to confirm BALF2-HR expression efficiency. The BALF2-HR overexpression group exhibited high levels of HLA-DP expression and reduced infiltration of CD8+ T cells, whereas the BALF2-HR knockout group displayed low HLA-DP expression with increased CD8+ T cell infiltration [Supplementary Fig.12G-I]. These findings suggest that BALF2-HR facilitates immune evasion in NPC cells in vivo and contributes to resistance against anti-PD-1 monotherapy. However, the combination of anti-LAG-3 and anti-PD-1 therapy was able to reverse BALF2-HR-mediated immune evasion, thereby enhancing the anti-tumor efficacy of anti-PD-1 treatment [Supplementary Fig.13].

Methods
Cell Lines
The nasopharyngeal carcinoma (NPC) cell lines HONE1, HK1, and the EBV-positive NPC cell line C666-1 used in this study were maintained in our laboratory at the Cancer Research Institute of Central South University. The HONE1-EBV and HK1-EBV cell lines were generously provided by Professor Tsao from the University of Hong Kong[71] and Professor Li from Southern Medical University[72]. Cells were cultured in RPMI 1640 medium (Gibco, 61870036, USA) supplemented with 10% fetal bovine serum (Vazyme Biotech Co.,Ltd, F103-01, China) and 1% penicillin-streptomycin (NCM Biotech, C100C5, China) at 37 ℃ with 5% CO2.

Clinical NPC Samples and recruited volunteers
The study involved three groups of clinical NPC tissue samples. The first group comprised 128 NPC tissues used for RNA extraction, quantitative real-time PCR (RT-qPCR), and subsequent Sanger sequencing to identify BALF2 subtypes (see Supplementary Data 1). The second group included 25 fresh NPC tissues prepared for single-cell suspensions and flow cytometry (see Supplementary Data 3). The third group consisted of paraffin-embedded sections from 58 NPC patients treated with PD-1 antibody, which were utilized for immunohistochemistry (IHC) and Sanger sequencing to identify BALF2 subtypes (see Supplementary Data 4). Human primary PBMCs were obtained from healthy volunteers. All uses of human material were confirmed by histopathological examination, and obtained with approval from The IRB of School of Basic Medical Sciences, Central South University or The IRB of Cancer Research Institute, Central South University and informed consents are obtained from patients and recruited volunteers. Additionally, the study utilized the GSE150825 database to analyze the differential expression of HLA-II molecules in four EBV-negative and six EBV-positive NPC tissues, and the GSE194333 database to compare HLA-II molecule expression between NP69 and C666-1 cell lines.

Immunohistochemistry (IHC)
IHC was performed using the Elivision plus (mouse/rabbit) IHC Kit (Fuzhou Maixin Biotech. Co., Ltd, KIT-9902, China). Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling in EDTA（Beyotime, P0084, China） buffer for 15 minutes. After washing with PBS（Servicebio, G0002, China）, endogenous peroxidase activity was blocked, and the sections were outlined with a hydrophobic pen. The sections were then blocked with goat serum, incubated overnight with primary antibodies, and subsequently incubated with a secondary antibody at room temperature for 10 minutes. Following this, the sections were treated with a biotin-peroxidase solution, developed with a DAB solution, counterstained with hematoxylin, dehydrated, and then mounted. Images were captured using an Olympus BX51 fluorescence microscope (Olympus, Japan). Staining intensity and positive rates were assessed independently by two experienced pathologists. Staining intensity was scored as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. Positive rates were scored as 0 = negative, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%.

Vectors, sgRNA, and Cell Transfection
The BALF2-LR and BALF2-HR overexpression vectors, along with sgBALF2, sgHLA-DP, sgHLA-DQ, and sgHLA-DR, were obtained from Beijing Tsingke Biotech Co., Ltd. The overexpression vectors for HLA-DP, HLA-DQ, and HLA-DR were constructed using the Pc3.1 vector. Transfection of the overexpression vectors, control plasmids, sgRNA, and negative controls into NPC cell lines was performed using the Neofect DNA transfection reagent (Neofect Biotech, Beijing, China). The sequence information for BALF2-LR and BALF2-HR is provided in Supplementary Data 6.

RNA Extraction and RT-qPCR
RNA was extracted from NPC cell lines using TRIzol reagent (Invitrogen™, 15596026CN,USA) and converted to cDNA with the HiScript cDNA Synthesis Kit (Vazyme, R223, China). Quantitative real-time PCR (RT-qPCR) was conducted in triplicate using SYBR Green (Vazyme, Q312, China), with GAPDH as the internal reference gene. Primer sequences are detailed in Supplementary Data 6.

Western blot
Total proteins were extracted from NPC cells using RISPA buffer (Yamaybio Inc, PC101, China) supplemented with protease inhibitors (Yamaybio Inc, GRF101, China) and phosphatase inhibitors (Beyotime Biotechnology, Shanghai, China). Proteins were separated by 10% ExpressCast PAGE (NCM Biotech, P2012, China) and transferred to a PVDF membrane (Vazyme, E801, China). Membranes were blocked and incubated overnight with primary antibodies, followed by a 1-hour incubation with secondary antibodies at room temperature. Protein bands were visualized using ECL detection reagent (Coolaber, SL1350, China), and images were captured with the Mini Chemiluminescent Imaging and Analysis System. Details of the antibodies used are provided in Supplementary Data 7.

Mass Spectrometry Analysis
BALF2-LR and BALF2-HR were overexpressed in the EBV-negative HK1 cell line, followed by Co-IP using an anti-BALF2 antibody. The precipitated proteins were analyzed via SDS-PAGE and high-throughput mass spectrometry (Jingjie PTM BioLab Co. Ltd) to identify interacting proteins. The mass spectrometry results are provided in Supplementary Data 5.

Co-Immunoprecipitation (Co-IP)
Transfected cells were collected 48 hours post-transfection and lysed using IP lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.1% SDS) supplemented with protease inhibitors (Keygen, KGB5106-1, China). A portion of the cell lysate was saved as an input control, while the remaining lysate was incubated with primary antibodies at room temperature for 2 hours. The lysates were then incubated with A/G beads (Selleck, B23202, China) at 4 ℃ overnight. After washing the beads, proteins were eluted by boiling in 95 ℃ SDS sample buffer and analyzed by Western blotting.

Bio-layer interferometry (BLI)
Bio-layer interferometry (BLI) was performed at 25 ℃ using the GatorPrime biosensor system (GatorBio) equipped with ProA probes. Fc-tagged LAG-3 (10 µg/ml) was immobilized onto the biosensors for 1 minute, followed by a 2-minute wash in PBS to establish the baseline. The biosensors were then immersed in wells containing varying concentrations of HLA-DP, HLA-DQ, or HLA-DR for 5 minutes to assess binding. Compound recombinant protein were purchased from MedChemExpress (Monmouth Junction, NJ, USA) This was followed by a 10-minute dissociation phase in PBS buffer. Data were analyzed using GraphPad Prism 9, applying a standard 1:1 binding model. Each sample was tested in two independent experiments.

Immunofluorescence
Transfected cells were plated in 24-well plates and fixed with 4% paraformaldehyde （Servicebio, G1101, China) for 15 minutes. After blocking with 5% BSA (Servicebio, GC305006, China) for 30 minutes, cells were incubated overnight with primary antibodies, followed by a 1-hour incubation with fluorescent secondary antibodies at room temperature. Nuclei were stained with DAPI (Invitrogen, D1306, USA) for 10 minutes. Images were captured using a confocal microscope (Ultra-View Vox, Perkin-Elmer, Waltham, MA, USA).

Flow Cytometry
Transfected tumor cells and CD8+T cells were co-cultured in 6-well plates at a 1:10 ratio for 24 hours. Tumor cell apoptosis was evaluated by collecting and digesting the co-cultured cells, followed by staining with an apoptosis detection kit (BD Pharmingen) and analysis using flow cytometry (BD LSRFortessa). To measure T cell cytokines, co-cultured CD8+T cells were treated with 10 µg/mL BFA (Selleck, S7046, China) for 6 hours, then stained with CD3 and CD8 antibodies, permeabilized, and further stained with fluorescently labeled cytokine antibodies. T cell exhaustion was assessed by staining co-cultured CD8+T cells with antibodies targeting CD3, CD8, PD-1, and TIM-3. Data analysis was conducted using FlowJo v10 software (Treestar). The flow gating strategy is shown in Supplementary Figures 4 and 13.

Enzyme-Linked Immunosorbent Assay (ELISA)
Supernatants from co-cultured tumor and CD8+T cells, as well as single-cell suspensions from tumors of nude mice, were collected after 24 hours and analyzed for cytokines using ELISA kits (Abcam, USA). Cytokine levels of IFN-γ, GZMB, and TNF were measured with a microplate reader equipped with SoftMax Pro 7 (Molecular Devices).

Crystal Violet Staining
Tumor cells cultured in 6-well plates were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% Crystal Violet solution (Coolaber, SL7081, China). The stained cells were scanned using a scanner, and then the stain was dissolved in 1 mL of DMSO (Coolaber, CD4731C, China). Absorbance was measured at 570 nm in 96-well plates.

Isolation of Human Primary T cells and DC
Peripheral blood from healthy volunteers was diluted 1:1 with saline and subjected to Ficoll-Hypaque (Cytiva, 17144003, USA) density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMCs). Monocytes were further enriched using CD14⁺ magnetic microbeads (Miltenyi Biotec, 130-050-201, Germany). The isolated monocytes were seeded at a density of 1.5 × 10⁶ cells/ml in 24-well plates and cultured for five days in complete RPMI 1640 medium supplemented with GM-CSF (100 ng/ml) and IL-4 (50 ng/ml). The purity of the resulting dendritic cells (DCs) was evaluated by flow cytometry.

Primary CD4⁺T cells (Miltenyi Biotec, 130-045-101, Germany) and CD8⁺T cells (Miltenyi Biotec, 130-045-201, Germany) were isolated from PBMCs using magnetic separation with CD4/CD8 microbeads. To generate NPC-reactive T cells, primary CD8⁺T cells from HLA-A–matched donors and primary CD4⁺T cells from HLA-DR–matched donors were co-cultured with autologous dendritic cells and irradiated NPC cells for one week [42, 51, 73, 74]. T cells were then activated and expanded using the T Cell Activation/Expansion Kit (Miltenyi Biotec, 130-091-441, Germany) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Umedium, 3022A, China), IL-2 (Novoprotein, Shanghai, C013, China) IL-7 (Novoprotein, Shanghai, C013, China), and IL-15 (Novoprotein, Shanghai, CX47, China) for 7–10 days. The purity of CD4⁺ and CD8⁺ T cell populations was verified by flow cytometry. All primary T cells used in the co-culture experiments were provided by 3 independent volunteers.

Animal Experiments
Two hundred female Balb/c Nude mice (4–6 weeks old, female, N-0007) were purchased from Silaikejingda Experimental Animal Co. (Hunan, China) and housed at the Animal Experiment Center of Hunan Cancer Hospital, in a Non-Specific Pathogen-Free (NSPF) environment. The experimental and control groups were housed separately to avoid any cross-contamination or bias. After preparation, HK1 cells transfected with BALF2-LR or BALF2-HR, and C666-1 cells transfected with sgBALF2, were subcutaneously injected into the right thigh inguinal region of the mice. Tumor formation was visually observed seven days after the initial treatment. Activated T cells were administered via tail vein injection. Some groups also received PD-1 antibody treatment or a combination of PD-1 and LAG-3 antibodies. Tumor volume was measured weekly. After 35 days for the HK1 cell lines and 42 days for the C666-1 cell lines, the mice were euthanized by cervical dislocation. Tumor tissues were excised, weighed, fixed, paraffin-embedded, and sectioned for immunohistochemistry. Single-cell suspensions from tumor tissues were prepared for flow cytometry to assess T cell function and exhaustion. Cytokine expression levels in tumor tissues were analyzed using ELISA and RT-qPCR.

To evaluate the role of BALF2-HR in promoting tumor immune evasion in vivo and the therapeutic efficacy of PD-1 and LAG-3 antibodies, 200 mice were randomized into 20 groups (10 mice per group). These groups included those injected with HK1 cell lines (Vector control, BALF2-LR overexpression, and BALF2-HR overexpression) and those injected with C666-1 cell lines (Negative control and BALF2 knockout). Additionally, the groups were divided based on treatment: five groups received activated T cell therapy seven days post-injection, five groups received activated T cell therapy with PD-1 antibody, and five groups received activated T cell therapy with the combination of PD-1 and LAG-3 antibodies. All animal experiments were approved by the Experimental Animal Ethics Committee of Central South University. The maximum tumor size permitted by the institutional ethical board was no more than 1,500mm³ and/or not exceeding 10% of the animal’s body weight. All experiments strictly adhered to these limits. Tumor size was measured using digital calipers, and tumor volume was calculated using the following formula: Tumor volume = 0.52×Length×Width2. Tumor measurements were performed two to three times per week. Animals were monitored daily for general health status, body weight, behavior, and signs of discomfort or distress. Criteria for early termination included: tumor volume exceeding the approved ethical limit, tumor ulceration or necrosis, rapid tumor growth affecting mobility or normal behavior, body weight loss greater than 15-20%, or any signs of severe distress such as lethargy, impaired movement, or abnormal posture.

Live-cell Imaging of T Cells
In vivo distribution of activated T cells in mice was evaluated using an in vivo imaging system (Bruker, USA). T cells were labeled with the deep red live-cell fluorescent dye DiR (ThermoFisher, D12731, China) prior to injection via the tail vein. Distribution was monitored at 1 hour, 1 day, 3 days, and 7 days post-injection under 3% isoflurane anesthesia (RWD, R510-22-16, China).

Statistical Analysis
All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad, USA). The Student’s t-test was employed to assess significant differences between two groups, while ANOVA was used for multiple comparisons. All data are presented as mean ± standard deviation (SD). A p-value of <0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The overall quality of paper is acceptable, and the concept is interesting.\nMy major concern is that since MHC-Class II is an important antigen presenting machinery against CD4 T cells, it is\ninteresting that only CD8 T cells were affected by high-risk EBV through BALF2. I understand that the authors have focused\non CD8 due to Fig.2A, but there should be some influence to CD4 if MHC-Class II is upregulated. Because there was no\nchanges in CD4 T cell population by Class II knockout in Fig. S8B, EBV- or NPC-reactive CD4 T cells may not be present in\nprimary CD4 T cells indicating that the use of primary T cells is not a best model to study tumor-reactive CD4 T cells. To\nconfirm that the finding in this study that high-risk EBV only affect CD8 T cells, EBV-specific or at least NPC-reactive CD4 T\ncells should be used in some experiments.", "reviewer_2": "The manuscript by Ge J. et al. describes an association of an endemic cancer, EBV-related nasopharyngeal carcinoma\n(NPC), with a variation in the Epstein-Barr virus (EBV) genome. There are several types of cancer that vary in incidence from\nregion to region. EBV-related malignancies are a representative type of cancer with regional differences, and the\ninvolvement of genetic predisposition, environmental and dietary differences, and EBV types has been described, although\nthe detailed mechanism(s) has not been clarified. The authors have addressed this issue by focusing on the variation in the\nEBV genome, 162215_C, 162476_C, and 163364_T, within the BALF2 gene, namely the high-risk BALF2 variant (BALF2-\nHR), and found that the BALF2-HR significantly upregulated the expression of host HLA class II molecules. Among the HLA\nclass II molecules, HLA-DP strongly bound to an immune checkpoint molecule, LAG-3, on CD8⁺ T cells and inhibited their\neffector functions, including the secretion of cytokines and cytotoxic molecules, leading to T cell exhaustion. Consequently,\nNPC cells evaded the antitumor immune response and compromised the efficacy of anti-PD-1 mAb therapy. In addition,\nBALF2-HR interacted with the nuclear transport protein KPNA2, resulting in the nuclear translocation of the class II\ntransactivator (CIITA), thereby enhancing the transcription of HLA-II molecules. Furthermore, the combination of anti-PD-1\nmAb and anti-LAG-3 mAb provided a far stronger antitumor activity compared to anti-PD-1 mAb alone.\nGeneral comments:\nIt is interesting to analyze the mechanism(s) of regional differences in oncogenic microbe-associated cancers. However, this\nstudy is descriptive, and no mechanistic insight was provided. In addition, the impact of this study is severely impaired since\nthere are critical problems with the experimental design and interpretation of the data, and several key points were not\nadequately addressed.\nFirst, there are many studies showing the importance of CD4-MHC class II-LAG-3 interactions (PMID: 30349037, PMID:\n39191259, etc.). However, the authors totally ignored the concept and provided only limited data for CD4+ T cells. Why were\nLAG-3 on CD8 + T cells mainly targeted by MHC class II expressed by NPC cells? Considering the future clinical\napplicaion, the target cells should be an important issue and needs to be clarified with detailed mechanism. Furthermore, the\nantigen presentation capacity of MHC class II expressed by NPC cells was not addressed in this study. Given the incidence\nrate associated with different HLAs, this needs to be clarified before concluding the importance of MHC class II-LAG-3\ninteractions.\nSecond is their immunological assays, particularly in vitro T cell assays. The authors employed an in vitro culture system to\ninvestigate T cell functions. In the assays, the authors stimulated PBMCs in a non-specific manner for 7-10 days using\nCD3/CD28 magnetic beads (as shown in Figure S5). Although the author described these as “primary T cells”, they are\n“artificially activated T cells”. Furthermore, these T cells were cultured with cancer cells without any consideration of MHC\nmismatches; therefore, considering the strong T cell stimulating ability of MHC mismatches, the T cell assays are not the\ntumor -specific T cell responses, but rather these T cell responses should be allo-specific T cell responses. The authors\nneed to clarify this point with antigen specificity. Otherwise, the data completely mislead the conclusions of this manuscript.\nFinally, the authors mention that the variation in the EBV genome, 162215_C, 162476_C, and 163364_T, within the BALF2\ngene was only observed in NPC cells. As this is a key point of this study, why this variation was only involved in NPC\ndevelopment, but not in other EBV-associated malignancies, including lymphoma, should be explored.\nOverall, this manuscript is immature in its current form and thus will not represent a major advance in the field.\nSpecific points:\n1. In many experiments, the authors used cell lines, such as HONE1 and HK cells, that overexpress the target molecules. In\nthis case, the expression levels must be carefully checked to avoid artificial misleading. An important example is the\nexpression of HLA molecules, HLA-DP, HLA-DQ and HLA-DR. The expression level of each molecule must be comparable.\nIn addition, co-immunoprecipitation assays should be performed using a primary cancer cell line, such as C666-1. More\nimportantly, the binding ability should be examined by Kd values.\n2. In Figure S1F, it is difficult to understand this clustering analysis. The detailed information in each cluster should be\nprovided.\n3. In Figure 2, the authors examined immune cells in the tumor microenvironment of NPCs. However, the detailed\nphenotypes of each immune cell population, including CD4+ T cells, were not examined. A more comprehensive analysis\nshould be performed. In addition, given the importance of LAG3 expression by CD8+ T cells in NPC development and\nprogression, the mechanism needs to be clarified.\n4. In Figure 3, the significance of HLA-DP-LAG-3 interactions is shown. Given the incidence rate associated with different\nHLAs, are there any differences in the interactions in different HLA-DP alleles? In this issue, clarifying the interaction lesion\nbetween HLA-DP-LAG-3 should strengthen their conclusion. Moreover, there are some cancers that express HLA class II\n(PMID: 30463850). Can HLA-DP alleles also inhibit CD8+ T cell responses in these cancers?\n5. In Figure S4, although the authors have provided a representative staining and gating method, the demarcation of each\ncell population, particularly CD4+ T cells, Treg cells, and cytokine-producing cells, is not clear. Control staining should be\nprovided.\n6. In Figure 4, after disrupting HLA-DP-LAG-3 interactions, why was anti-PD-1 + anti-LAG-3 superior to anti-PD-1 alone? Are\nthere any other mechanism(s) to induce LAG-3 suppression?\n7. In Figure 5E and S11G-H, the authors show immunohistochemistry images. The poor quality of these images makes them\ninterpret with great difficulties. More importantly, the background staining is strong, which hinders the proper understanding\nof these images. In addition, the size markers are difficult to see.\n8. Most of the Discussion section should be moved to the Introduction section, such as lines 462-531, which describe\ngeneral knowledge.", "reviewer_3": "• General Comments:\nThis manuscript presents an in-depth review of EBV and its role in cancer, particularly its interactions with the tumor\nmicroenvironment and immune evasion mechanisms.\nSpecifically, it identifies the BALF2-HR variant in NPC cells to significantly upregulate HLA-DP that binds to immune\ncheckpoint molecule LAG-3 on the T cell surface and suppresses the secretion of cytotoxic cytokines, promoting T cell\nexhaustion. Suggestively, the use of LAG-3 and PD-1 monoclonal antibodies significantly improves the efficacy of\nimmunotherapy in NPC.\nThe topic is highly relevant to the field of viral oncology and immunotherapy, particularly in regionally associated high-risk\nEBV subtype infections. The manuscript is well-structured and provides a comprehensive overview of recent advances\nwhile providing a novel perspective supporting the application of combinational immune checkpoint inhibitor therapy for\npatients infected with high-risk EBV subtypes. However, there are several concerns regarding clarity and scientific rigor that\nshould be addressed.\n• Specific Comments:\n- The introduction should provide a more concise and focused background on EBV's oncogenic mechanisms before diving\ninto specific interactions within the tumor microenvironment.\n- Lines 66/67, the authors state that “NPC displays a significant geographic concentration in sourthern China.” Please add a\nreference to back this claim.\n- The study identified the C-C-T high-risk EBV genotype in 124 patients, establishing its association with NPC. However,\nhas this C-C-T subtype also been detected in EBV-positive healthy controls within the region? Including the authors'\nperspective on this would enhance the manuscript's value.\n- The biological effects of polymorphisms in the EBV BALF2 gene have been demonstrated in previous studies. (Eg.: Chee\nYit Lim, Gwyneth W.Y. Ng, Chuan Keng Goh, Melvin Kiang Chua Lee, Ian Cheong, Eng Eong Ooi, Jianjun Liu, Robert B.\nWest, Kwok Seng Loh, Joshua K. Tay, Impact of high-risk EBV strains on nasopharyngeal carcinoma gene expression, Oral\nOncology.) Therefore, it would be beneficial to connect this study's findings to the broader literature and highlight relevant\nparallels.\n- PD-1 and LAG-3 expression have been shown to impact patient survival in other EBV-associated cancers. (Eg.: Jimenez\nO, Mangiaterra T, Colli S, García Lombardi M, Preciado MV, De Matteo E, Chabay P. PD-1 and LAG-3 expression in EBV-\nassociated pediatric Hodgkin lymphoma has influence on survival. Frontiers Oncology) Drawing parallels to indicate similar\neffects in EBV-associated NPC would strengthen the manuscript's discussion.\n- The authors suggest that the BALF2-HR variant may be specific to regionally associated NPC cases in Southern China. It\nwould be beneficial to discuss how the findings of this study—particularly the potential of combinational anti-LAG-3 and PD-\n1 therapy for NPC patients infected with high-risk EBV subtypes—could be extrapolated to other regionally associated NPC\ncases. This includes considering the implications for patients affected by the global high-risk EBV variant and the North\nAfrican high-risk EBV variant.\nThe discussion section contains an extended narrative covering well-established facts about EBV and NPC. This can be\nstreamlined by focusing only on details directly relevant to the section and the study's findings.\n- A dedicated \"Future Directions\" paragraph summarizing key questions and potential research avenues would enhance the\nmanuscript’s impact.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Answer to the Reviewers\nReviewer #1 (Remarks to the Author):\nThe remaining figures could also be reduced to the most important panels. The overall\nquality of paper is acceptable, and the concept is interesting.\nMy major concern is that since MHC-Class II is an important antigen presenting\nmachinery against CD4 T cells, it is interesting that only CD8 T cells were affected by\nhigh-risk EBV through BALF2. I understand that the authors have focused on CD8 due to\nFig.2A, but there should be some influence to CD4 if MHC-Class II is upregulated.\nBecause there was no changes in CD4 T cell population by Class II knockout in Fig. S8B,\nEBV- or NPC-reactive CD4 T cells may not be present in primary CD4 T cells indicating\nthat the use of primary T cells is not a best model to study tumor-reactive CD4 T cells. To\nconfirm that the finding in this study that high-risk EBV only affect CD8 T cells, EBV-\nspecific or at least NPC-reactive CD4 T cells should be used in some experiments.\nResponse: Thank you for your thoughtful suggestions. To assess whether BALF2\nencoded by high-risk EBV modulates CD4⁺ T cell function, we first isolated PBMCs\nfrom human peripheral blood and used CD4 microbeads to purify primary CD4⁺ T cells.\nA subset of these cells was co-cultured for one week with irradiated HK1 or C666-1 cells\nto generate NPC-reactive CD4⁺ T cells. All primary T cells used in this study were\nisolated from donors with HLA-A alleles matched to those of the tumor cells, ensuring\nantigen compatibility. We then co-cultured NPC cells with either primary CD4⁺ T cells or\nNPC-reactive CD4⁺ T cells. Flow cytometry analysis showed that IL-2, Ki67, and CD69\nexpression was significantly upregulated in NPC-reactive CD4⁺ T cells, while no notable\nchanges were observed in primary CD4⁺ T cells [Fig. S4E].\nNext, BALF2-HR or BALF2-LR was overexpressed in HK1 and HONE1 cells, and\nBALF2-HR was knocked out in C666-1 cells. These tumor cells were co-cultured with\nNPC-reactive CD4⁺ T cells, and the expression of IL-2, Ki67, CD69, and FOXP3 in\nCD4⁺ T cells was assessed by flow cytometry. The results indicated that BALF2-HR\nexerted only a mild regulatory effect on NPC-reactive CD4⁺ T cells [Fig. S4F]. Because\neffective CD4⁺ T cell activation requires both TCR recognition of HLA-II–peptide\ncomplexes (signal 1) and engagement of co-stimulatory molecules (signal 2, e.g.,\nCD80/CD86 binding to CD28), we analyzed single-cell RNA-seq data from the\nGSE150430 dataset. This analysis revealed that CD80/CD86 were highly expressed on\ndendritic cells but minimally expressed on tumor cells [Fig. S4G]. Therefore, we believe\nthe main reason CD4+ T cells are not fully activated by tumor cells is the lack of co-\nstimulatory signals from these cells.\nTo identify the primary T cell subsets targeted by BALF2-HR in nasopharyngeal\ncarcinoma (NPC), primary CD8⁺ T cells were isolated from human PBMCs using CD8\nmicrobeads. These cells were then co-cultured with mature dendritic cells and irradiated\nNPC cells for one week to generate NPC-reactive CD8⁺ T cells. Subsequently, NPC-\nreactive CD4⁺ T cells, NPC-reactive CD8⁺ T cells, or a combination of both were co-\ncultured with tumor cells overexpressing BALF2-HR. Tumor cell viability, assessed by\ncrystal violet staining, revealed that BALF2-HR modulates tumor cell responses\nprimarily through CD8⁺ T cells rather than CD4⁺ T cells [Fig. S4H]. Flow cytometry\nanalysis of tumor cell apoptosis further supported this finding [Fig. S4I]. Single-cell RNA\nsequencing analysis of the GSE150430 dataset showed that the inhibitory receptor LAG-\n3 is predominantly expressed on CD8⁺ T cells, with comparatively low expression on\nCD4⁺ T cells [Fig. S4J]. Together, these results indicate that BALF2-HR primarily targets\nCD8⁺ T cells, promoting tumor immune evasion largely by driving CD8⁺ T cell\nexhaustion.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Ge J. et al. describes an association of an endemic cancer, EBV-\nrelated nasopharyngeal carcinoma (NPC), with a variation in the Epstein-Barr virus\n(EBV) genome. There are several types of cancer that vary in incidence from region to\nregion. EBV-related malignancies are a representative type of cancer with regional\ndifferences, and the involvement of genetic predisposition, environmental and dietary\ndifferences, and EBV types has been described, although the detailed mechanism(s) has\nnot been clarified. The authors have addressed this issue by focusing on the variation in\nthe EBV genome, 162215_C, 162476_C, and 163364_T, within the BALF2 gene, namely\nthe high-risk BALF2 variant (BALF2-HR), and found that the BALF2-HR significantly\nupregulated the expression of host HLA class II molecules. Among the HLA class II\nmolecules, HLA-DP strongly bound to an immune checkpoint molecule, LAG-3, on\nCD8⁺ T cells and inhibited their effector functions, including the secretion of cytokines\nand cytotoxic molecules, leading to T cell exhaustion. Consequently, NPC cells evaded\nthe antitumor immune response and compromised the efficacy of anti-PD-1 mAb therapy.\nIn addition, BALF2-HR interacted with the nuclear transport protein KPNA2, resulting in\nthe nuclear translocation of the class II transactivator (CIITA), thereby enhancing the\ntranscription of HLA-II molecules. Furthermore, the combination of anti-PD-1 mAb and\nanti-LAG-3 mAb provided a far stronger antitumor activity compared to anti-PD-1 mAb\nalone.\nGeneral comments:\nIt is interesting to analyze the mechanism(s) of regional differences in oncogenic\nmicrobe-associated cancers. However, this study is descriptive, and no mechanistic\ninsight was provided. In addition, the impact of this study is severely impaired since there\nare critical problems with the experimental design and interpretation of the data, and\nseveral key points were not adequately addressed.\nResponse: Thank you for your thoughtful review of our manuscript. Your valuable\nfeedback has significantly enhanced our work during the revision process. In this revised\nversion, we have addressed your critiques carefully and included a substantial amount of\nadditional experimental data to support our conclusions. We believe that the manuscript\nis now greatly improved compared to the original version we submitted. We appreciate\nyour guidance.\nFirst, there are many studies showing the importance of CD4-MHC class II-LAG-3\ninteractions (PMID: 30349037, PMID: 39191259, etc.). However, the authors totally\nignored the concept and provided only limited data for CD4+ T cells. Why were\nLAG-3 on CD8 + T cells mainly targeted by MHC class II expressed by NPC cells? Considering\nthe future clinical applicaion, the target cells should be an important issue and needs to be\nclarified with detailed mechanism. Furthermore, the antigen presentation capacity of\nMHC class II expressed by NPC cells was not addressed in this study. Given the\nincidence rate associated with different HLAs, this needs to be clarified before\nconcluding the importance of MHC class II-LAG-3 interactions.\nResponse: Thank you for raising these important concerns. To investigate whether\nBALF2, encoded by high-risk EBV, regulates CD4⁺ T cell function, we first isolated\nhuman peripheral blood PBMCs and purified primary CD4⁺ T cells using CD4\nmicrobeads. A subset of these primary CD4⁺ T cells was co-cultured for one week with\nirradiated HK1 or C666-1 cells to generate NPC-reactive CD4⁺ T cells. Following the\nsuggestions from you and Reviewer 1, we established and validated this NPC-reactive\nCD4⁺ T cell model and co-cultured the cells with nasopharyngeal carcinoma cells. We\nfound that BALF2-HR and HLA class II molecules had only a modest regulatory effect\non the expression of IL-2, CD69, Ki67, and FOXP3 in CD4⁺ T cells [Fig. S4F, Fig. S9A].\nEffective activation of CD4⁺ T cells require not only TCR-specific recognition of the\nHLA-II–antigen peptide complex (signal 1) but also engagement of co-stimulatory\nmolecules (such as CD80/CD86) on CD4⁺ T cells with CD28 (signal 2). Analysis of\nsingle-cell sequencing data from GSE150430 showed that CD80/CD86 were highly\nexpressed mainly on dendritic cells, but were scarcely expressed on tumor cells [Fig.\nS4G]. Therefore, we believe the main reason CD4+ T cells are not fully activated by\ntumor cells is the lack of co-stimulatory signals from these cells.\nTo investigate the primary T cell subsets targeted by BALF2-HR in NPC, we isolated\nprimary CD8⁺ T cells from human peripheral blood mononuclear cells (PBMCs) using\nCD8 microbeads. These CD8⁺ T cells were then co-cultured with dendritic cells and\nirradiated nasopharyngeal carcinoma cells for one week to generate NPC-reactive CD8⁺ T\ncells. Subsequently, NPC-reactive CD4⁺ T cells, NPC-reactive CD8⁺ T cells, or a\ncombination of both were co-cultured with tumor cells expressing BALF2-HR. Crystal\nviolet staining assessing tumor cell viability revealed that BALF2-HR modulates tumor\ncell function primarily through CD8⁺ T cells, with little effect via CD4⁺ T cells [Fig.\nS4H]. Flow cytometry analysis of tumor cell apoptosis corroborated these results [Fig.\nS4I]. Further analysis of single-cell RNA sequencing data from the GSE150430 dataset\ndemonstrated that LAG-3 is predominantly expressed on CD8⁺ T cells, while its\nexpression on CD4⁺ T cells is relatively low [Fig. S4J]. Together, these findings suggest\nthat BALF2-HR mainly targets CD8⁺ T cells and promotes tumor immune evasion\nprimarily by inducing CD8⁺ T cell exhaustion.\nSecond is their immunological assays, particularly in vitro T cell assays. The authors\nemployed an in vitro culture system to investigate T cell functions. In the assays, the\nauthors stimulated PBMCs in a non-specific manner for 7-10 days using CD3/CD28\nmagnetic beads (as shown in Figure S5). Although the author described these as “primary\nT cells”, they are “artificially activated T cells”. Furthermore, these T cells were cultured\nwith cancer cells without any consideration of MHC mismatches; therefore, considering\nthe strong T cell stimulating ability of MHC mismatches, the T cell assays are not the\ntumor -specific T cell responses, but rather these T cell responses should be allo-specific\nT cell responses. The authors need to clarify this point with antigen specificity. Otherwise,\nthe data completely mislead the conclusions of this manuscript.\nResponse: Thanks for your critical suggestions. We apologize for the lack of clarity in\nthe flowchart and the Materials and Methods section. Prior to co-culturing T cells with\ntumor cells, we perform HLA-A typing of both the volunteers and tumor cells to ensure\nmatching with the corresponding nasopharyngeal carcinoma cells (PMID: 35165282,\nPMID: 39366979). Before co-culture, primary T cells are incubated with irradiated,\ninactivated tumor cells and mature dendritic cells for 7 days to induce a tumor-specific T\ncell response (PMID: 39542656). This protocol was detailed in our previous publication,\nwhich led to its omission from the current Materials and Methods section. We apologize\nfor this oversight and have now included additional details accordingly.\nFinally, the authors mention that the variation in the EBV genome, 162215_C, 162476_C,\nand 163364_T, within the BALF2 gene was only observed in NPC cells. As this is a key\npoint of this study, why this variation was only involved in NPC development, but not in\nother EBV-associated malignancies, including lymphoma, should be explored. Overall,\nthis manuscript is immature in its current form and thus will not represent a major\nadvance in the field.\nResponse: Thanks for your highly relevant concern. In NPC, EBV predominantly\nestablishes latency type II and relies on certain lytic genes, such as BALF2, to sustain\nviral replication. A distinct feature of NPC is the occurrence of an abortive lytic cycle, in\nwhich lytic-phase proteins are expressed in many EBV-positive tumor cells without\nleading to cell lysis. In contrast, lymphomas such as Burkitt’s lymphoma typically exhibit\nlatency type I, characterized by highly stable EBV genomes and suppressed expression of\nlytic genes, including BALF2 (PMID: 38206974).\nSpecific points:\n1. In many experiments, the authors used cell lines, such as HONE1 and HK cells, that\noverexpress the target molecules. In this case, the expression levels must be carefully\nchecked to avoid artificial misleading. An important example is the expression of HLA\nmolecules, HLA-DP, HLA-DQ and HLA-DR. The expression level of each molecule\nmust be comparable. In addition, co-immunoprecipitation assays should be performed\nusing a primary cancer cell line, such as C666-1. More importantly, the binding ability\nshould be examined by Kd values.\nResponse: Thanks for these excellent suggestions. We revised the experimental design\nand re-evaluated the overexpression efficiency of HLA-DP, HLA-DQ, and HLA-DR at\nboth the mRNA and protein levels using RT-qPCR and Western blot analysis [Fig. S6].\nSubsequently, co-immunoprecipitation (Co-IP) assays were performed in the EBV-\npositive NPC cell line C666-1 to confirm the physical interaction between BALF2 and\nKPNA2 [Fig. S11C]. Finally, to assess the binding affinity between LAG-3 and HLA\nclass II molecules, recombinant proteins of HLA-DP, HLA-DQ, and HLA-DR were\nindividually co-incubated with recombinant LAG-3 protein. Bio-layer interferometry\n(BLI) binding studies revealed that HLA-DP binds LAG-3 with significantly higher\naffinity (Kd = 0.164 nM) compared to HLA-DQ (Kd = 28.4 nM) and HLA-DR (Kd =\n23.8 nM) [Fig. 5A, Fig. S10A].\n2. In Figure S1F, it is difficult to understand this clustering analysis. The detailed\ninformation in each cluster should be provided.\nResponse: Thanks for your kind suggestions. We have provided additional detailed\nannotations for each cluster [Fig. S1F].\n3. In Figure 2, the authors examined immune cells in the tumor microenvironment of\nNPCs. However, the detailed phenotypes of each immune cell population, including\nCD4+ T cells, were not examined. A more comprehensive analysis should be performed.\nIn addition, given the importance of LAG3 expression by CD8+ T cells in NPC\ndevelopment and progression, the mechanism needs to be clarified.\nResponse: Thanks for your suggestions. We have provided detailed phenotypic analyses\nof CD4⁺ T cells in 16 NPC tissue samples infected with high-risk EBV subtypes and 9\nsamples with low-risk subtypes using flow cytometry. This included assessment of\nmarkers for Treg (FOXP3), Th1 (T-bet), Th2 (GATA3), and Th17 (RORγT) subsets. The\nresults showed no significant differences in the frequencies of total CD4⁺ T cells, Treg\ncells, Th1 cells, Th2 cells, or Th17 cells between high-risk and low-risk EBV-infected\nNPC tissues [Fig. S4C–D].\nTo further elucidate the molecular mechanism by which LAG-3 inhibits T cell function,\nwe focused on its activation via ubiquitination—a critical post-translational modification\nrequired for LAG-3 to exert its immunosuppressive effects (PMID: 40101708). We\noverexpressed BALF2-HR in the NPC cell lines HONE1 and HK1, or knocked out\nBALF2-HR in C666-1 cells, and subsequently co-cultured these cells with CD8⁺ T cells.\nParallel cultures were treated with either IgG or a LAG-3 monoclonal antibody (mAb).\nCo-immunoprecipitation (Co-IP) assays revealed that BALF2-HR overexpression\nsignificantly enhanced LAG-3 ubiquitination in CD8⁺ T cells, whereas BALF2-HR\nknockout markedly reduced this modification. Notably, treatment with LAG-3 mAb\nblocked the BALF2-HR-induced increase in LAG-3 ubiquitination [Fig. 5C]. Lck\n(lymphocyte-specific protein tyrosine kinase) plays a pivotal role in T cell receptor (TCR)\nsignaling. Previous studies have shown that LAG-3 can interfere with the interaction\nbetween CD8 and Lck (PMID: 40592325, PMID: 35437325). Co-IP assays demonstrated\nthat BALF2-HR significantly disrupted CD8–Lck binding, thereby attenuating\ndownstream TCR signaling. This inhibitory effect was reversed by the addition of LAG-3\nmAb [Fig. 5D]. These findings indicate that BALF2-HR disrupts the interaction between\nCD8 and Lck by activating the ubiquitination of LAG-3, which may lead to further\ninhibition of downstream TCR signaling.\nPhosphorylation of ZAP70 acts as a \"molecular switch\" in TCR signal transduction,\nwhile LAG-3 has been shown to promote the expression of the exhaustion-associated\ntranscription factor TOX (PMID: 39121848). Western blot analysis revealed that BALF2-\nHR expression in tumor cells reduced ZAP70 phosphorylation and elevated TOX levels\nin CD8⁺ T cells. Notably, treatment with a LAG-3 monoclonal antibody (mAb) restored\nZAP70 phosphorylation and suppressed TOX expression [Fig. 5E]. Collectively, these\nfindings demonstrate that BALF2-HR activates LAG-3 via ubiquitination, thereby\ndisrupting CD8–Lck interactions and attenuating downstream TCR signaling through\nreduced ZAP70 phosphorylation. This signaling impairment leads to TOX upregulation\nand contributes to CD8⁺ T cell exhaustion. These results provide a mechanistic rationale\nfor combining LAG-3 and PD-1 blockade in the immunotherapy of EBV-associated NPC.\n4. In Figure 3, the significance of HLA-DP-LAG-3 interactions is shown. Given the\nincidence rate associated with different HLAs, are there any differences in the\ninteractions in different HLA-DP alleles? In this issue, clarifying the interaction lesion\nbetween HLA-DP-LAG-3 should strengthen their conclusion. Moreover, there are some\ncancers that express HLA class II (PMID: 30463850). Can HLA-DP alleles also inhibit\nCD8+ T cell responses in these cancers?\nResponse: Thanks for your critical reading. In the present study, we used the NCBI\nreference sequences for HLA-DP, specifically HLA-DPA101:03 and HLA-DPB104:01.\nGiven the population-specific distribution of HLA-DP alleles, deep sequencing of the\nMHC-II region in the Chinese population revealed that the most prevalent HLA-DPA1\nalleles are *02:02 (48.54%), *01:03 (39.39%), and *02:01 (11.35%), while the dominant\nHLA-DPB1 alleles are *05:01 (36.86%), *02:01 (19.74%), and *04:01 (10.60%) (PMID:\n27213287). To explore the functional differences among HLA-DP alleles, we synthesized\ncorresponding plasmids and overexpressed these variants in HK1 and C666-1\nnasopharyngeal carcinoma cell lines. Overexpression of the various alleles did not\nsignificantly affect tumor cell proliferation [Fig. S7A-B]. However, when these tumor\ncells were co-cultured with human primary T cells, crystal violet staining assays showed\nthat different HLA-DP alleles enhanced tumor cell survival to varying degrees [Fig.\nS7C], with flow cytometry analysis of apoptosis yielding consistent results [Fig. S7D].\nFurther flow cytometric analysis revealed that the different HLA-DP alleles variably\nsuppressed cytotoxic cytokine production (e.g., IFN-γ, TNF-α) in CD8⁺ T cells and\npromoted their exhaustion [Fig. S7E-F]. Collectively, these findings suggest that HLA-\nDP alleles may contribute to immune evasion in nasopharyngeal carcinoma by\ndifferentially modulating CD8⁺ T cell function—potentially via allele-specific amino acid\ndifferences that alter the structure of the antigen-binding groove and its interaction with\nLAG-3.\nIn addition, certain cancers have been reported to express HLA class II molecules\n(PMID: 30463850). Given the highly conserved specificity of LAG-3 binding to HLA-\nDP—exhibiting markedly stronger affinity than to HLA-DQ or HLA-DR—our findings\nsuggest that in immunologically active (\"hot\") tumors, the expression of HLA class II\nmolecules such as HLA-DP on tumor cells may suppress CD8⁺ T cell-mediated anti-\ntumor responses via LAG-3–dependent inhibitory signaling. This study provides new\ninsights into the immunomodulatory role of HLA class II molecules in tumor cells.\nFuture investigations into other HLA class II–expressing cancers may further inform the\ndevelopment of effective immunotherapeutic strategies.\n5. In Figure S4, although the authors have provided a representative staining and gating\nmethod, the demarcation of each cell population, particularly CD4+ T cells, Treg cells,\nand cytokine-producing cells, is not clear. Control staining should be provided.\nResponse: Thanks for your helpful suggestions. We have supplemented the control\nstaining for the flow cytometry experiment (Fluorescence Minus One [FMO] control)\n[Fig. S4B].\n6. In Figure 4, after disrupting HLA-DP-LAG-3 interactions, why was anti-PD-1 + anti-\nLAG-3 superior to anti-PD-1 alone? Are there any other mechanism(s) to induce LAG-3\nsuppression?\nResponse: Thank you for raising these important points. The immunosuppressive\nfunction of LAG-3 is not solely dependent on HLA-II molecules. Ligands such as\nGalectin-3 (Gal-3), fibrinogen-like protein 1 (FGL1), and liver sinusoidal endothelial cell\nlectin (LSECtin) provide MHC-II–independent binding pathways for LAG-3,\ncontributing to its inhibitory effects (PMID: 37488429). Consequently, even after the\nHLA-DP knockout, combination therapy using PD-1 and LAG-3 monoclonal antibodies\n(mAbs) is still more effective than PD-1 monotherapy.\n7. In Figure 5E and S11G-H, the authors show immunohistochemistry images. The poor\nquality of these images makes them interpret with great difficulties. More importantly,\nthe background staining is strong, which hinders the proper understanding of these\nimages. In addition, the size markers are difficult to see.\nResponse: We appreciate your valuable suggestions. We repeated the IHC experiments\nand included improved representative image results [Fig.5E, Fig. S11G-H].\n8. Most of the Discussion section should be moved to the Introduction section, such as\nlines 462-531, which describe general knowledge.\nResponse: Thank you for your helpful suggestions. We have relocated some of the\ndiscussion content to the Introduction section.\nReviewer #3 (Remarks to the Author):\n• General Comments:\nThis manuscript presents an in-depth review of EBV and its role in cancer, particularly its\ninteractions with the tumor microenvironment and immune evasion mechanisms.\nSpecifically, it identifies the BALF2-HR variant in NPC cells to significantly upregulate\nHLA-DP that binds to immune checkpoint molecule LAG-3 on the T cell surface and\nsuppresses the secretion of cytotoxic cytokines, promoting T cell exhaustion.\nPage 10 of 13\nSuggestively, the use of LAG-3 and PD-1 monoclonal antibodies significantly improves\nthe efficacy of immunotherapy in NPC.\nThe topic is highly relevant to the field of viral oncology and immunotherapy,\nparticularly in regionally associated high-risk EBV subtype infections. The manuscript is\nwell-structured and provides a comprehensive overview of recent advances while\nproviding a novel perspective supporting the application of combinational immune\ncheckpoint inhibitor therapy for patients infected with high-risk EBV subtypes. However,\nthere are several concerns regarding clarity and scientific rigor that should be addressed.\nResponse: We appreciate the reviewer’s positive feedback on our manuscript. Thank you\nfor your encouraging comments.\n• Specific Comments:\n- The introduction should provide a more concise and focused background on EBV's\noncogenic mechanisms before diving into specific interactions within the tumor\nmicroenvironment.\nResponse: We appreciate your valuable suggestions and have provided a concise and\nfocused overview of EBV's oncogenic mechanisms in the Introduction Section. Lines 56-\n59.\n- Lines 66/67, the authors state that “NPC displays a significant geographic concentration\nin sourthern China.” Please add a reference to back this claim.\nResponse: Thanks for your suggestion. We have included the references to back the\nclaim (PMID: 34699681, PMID: 38359790).\n- The study identified the C-C-T high-risk EBV genotype in 124 patients, establishing its\nassociation with NPC. However, has this C-C-T subtype also been detected in EBV-\npositive healthy controls within the region? Including the authors' perspective on this\nwould enhance the manuscript's value.\nResponse: Thanks for pointing this out. In our research group, saliva samples were\ncollected from 20 healthy individuals. DNA was extracted using the AxyPrep Body Fluid\nViral DNA/RNA Miniprep Kit according to the manufacturer’s instructions. EBV DNA\nwas not detected in any of the 20 samples. Although approximately 90%–95% of adults\nPage 11 of 13\nworldwide are seropositive for EBV, the virus typically persists in a latent, asymptomatic\nstate in healthy individuals. Active replication of EBV in saliva occurs only in a small\nsubset of cases, leading to a much lower EBV DNA detection rate in saliva compared to\nseropositivity (PMID: 27687982, PMID: 27687982). Importantly, the presence of active\nEBV replication in saliva may serve as an early warning indicator for NPC.\n- The biological effects of polymorphisms in the EBV BALF2 gene have been\ndemonstrated in previous studies. (Eg.: Chee Yit Lim, Gwyneth W.Y. Ng, Chuan Keng\nGoh, Melvin Kiang Chua Lee, Ian Cheong, Eng Eong Ooi, Jianjun Liu, Robert B. West,\nKwok Seng Loh, Joshua K. Tay, Impact of high-risk EBV strains on nasopharyngeal\ncarcinoma gene expression, Oral Oncology.) Therefore, it would be beneficial to connect\nthis study's findings to the broader literature and highlight relevant parallels.\nResponse: Thanks for your suggestion. Following the reviewers’ suggestions, we have\ndiscussed the findings of this study in relation to this reference in the Discussion section\n(lines 545–551).\n- PD-1 and LAG-3 expression have been shown to impact patient survival in other EBV-\nassociated cancers. (Eg.: Jimenez O, Mangiaterra T, Colli S, García Lombardi M,\nPreciado MV, De Matteo E, Chabay P. PD-1 and LAG-3 expression in EBV-associated\npediatric Hodgkin lymphoma has influence on survival. Frontiers Oncology) Drawing\nparallels to indicate similar effects in EBV-associated NPC would strengthen the\nmanuscript's discussion.\nResponse: Thanks for your thoughtful suggestion. In response to your comments, we\nhave added relevant discussion in lines 601–603.\n.- The authors suggest that the BALF2-HR variant may be specific to regionally\nassociated NPC cases in Southern China. It would be beneficial to discuss how the\nfindings of this study—particularly the potential of combinational anti-LAG-3 and PD-1\ntherapy for NPC patients infected with high-risk EBV subtypes—could be extrapolated to\nother regionally associated NPC cases. This includes considering the implications for\npatients affected by the global high-risk EBV variant and the North African high-risk\nEBV variant.\nPage 12 of 13\nResponse: Thank you for your suggestion. We have had a discussion based on your\ncomment on this issue. Lines 612-614.\n- The discussion section contains an extended narrative covering well-established facts\nabout EBV and NPC. This can be streamlined by focusing only on details directly\nrelevant to the section and the study's findings.\nResponse: Thank you for your valuable suggestion. We have revised the Discussion\nsection in accordance with your feedback.\n- A dedicated \"Future Directions\" paragraph summarizing key questions and potential\nresearch avenues would enhance the manuscript’s impact.\nResponse: Thanks for the suggestion. we have now added a 'Future Research Directions'\nparagraph to the Discussion section (lines 631–643).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We appreciate your suggestions and those of the other reviewers.\nPage 13 of 13"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have successfully addressed my previous concerns.", "reviewer_2": "In this manuscript by Ge J. et al., the authors mostly answered to the concerns I raised. However, some points were not adequately addressed.\n1. This reviewer was not persuaded by their T cell assays. While the authors explain that “Prior to co-culturing T cells with tumor cells, we perform HLA-A typing of both the volunteers and tumor cells to ensure matching with the corresponding nasopharyngeal carcinoma cells”, MHC class II mismatches were ignored. This could still result in allo-specific T cell responses, though the authors surprisingly did not observe such responses. Additionally, since cancer cells can directly stimulate CD4+ T cells (PMID: 35508657), their claim that signal 2's absence failed to stimulate CD4+ T cells is insufficient to rule out potential interactions between CD4+ T cells and cancer cells. The authors must address this issue more carefully to strengthen their conclusion. For example, the authors provided TCR signaling data with/without anti-LAG3 mAb in Figure 5E. This should also be assessed using CD4+ T cells.\nFurthermore, as the authors discussed in response to my query #6, since “the immunosuppressive function of LAG-3 is not solely dependent on HLA-II molecules”, this new mechanism of LAG3 immunosuppression is one addition to the previous findings. This needs to be clarified in the discussion section.\n2. The variation in the EBV genome in NPC cells attributed to latency type II is plausible. However, extranodal NK/T cell lymphoma and Hodgkin lymphoma are also latency type II malignancies (PMID: 26449716). Therefore, the authors' explanation is insufficient to account for the variation in the EBV genome in NPC cells.\n3. Regarding the clustering analysis in Figure S1F, information on the marker genes used to identify each cluster and their expression levels should be provided.", "reviewer_3": "I have reviewed the authors’ responses to my comments. All of my questions and concerns have been addressed appropriately. The missing references I requested have also been added.\nI have no further comments at this stage.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Answer to the Reviewers\nReviewer #1 (Remarks to the Author):\nThe authors have successfully addressed my previous concerns.\nResponse: Thank you very much!\nReviewer #2 (Remarks to the Author):\nIn this manuscript by Ge J. et al., the authors mostly answered to the concerns I raised.\nHowever, some points were not adequately addressed.\n1. This reviewer was not persuaded by their T cell assays. While the authors explain\nthat “Prior to co-culturing T cells with tumor cells, we perform HLA-A typing of both\nthe volunteers and tumor cells to ensure matching with the corresponding\nnasopharyngeal carcinoma cells”, MHC class II mismatches were ignored. This could\nstill result in allo-specific T cell responses, though the authors surprisingly did not\nobserve such responses. Additionally, since cancer cells can directly stimulate CD4+ T\ncells (PMID: 35508657), their claim that signal 2's absence failed to stimulate CD4+ T\ncells is insufficient to rule out potential interactions between CD4+ T cells and cancer\ncells. The authors must address this issue more carefully to strengthen their conclusion.\nFor example, the authors provided TCR signaling data with/without anti-LAG3 mAb\nin Figure 5E. This should also be assessed using CD4+ T cells.\nResponse：Thank you for your detailed review and valuable comments on our\nmanuscript. Our study aims to investigate how the high-risk EBV BALF2 protein\npromotes immune evasion in nasopharyngeal carcinoma by upregulating MHC class II\nmolecules during carcinogenesis. In actual nasopharyngeal carcinoma patients, MHC\nclass II molecule mismatch does not occur, which is why we overlooked the potential\nissue of MHC class II mismatch that you mentioned.\nDue to the lack of murine nasopharyngeal carcinoma cell lines and in situ-induced\nnasopharyngeal carcinoma mouse models, as well as the absence of EBV receptors in\nmice (which means EBV cannot infect mice), we were limited to using nasopharyngeal\ncarcinoma cell lines and T cells derived from healthy volunteers for in vitro co-culture\nexperiments. Following your suggestion, we repeated the tumor cell and T cell co-\nculture experiments, especially with CD4+ T cells, and confirmed that when tumor cells\nand CD4+ T cells have mismatched MHC class II molecules, it indeed causes severe\nimmune rejection.\nTherefore, we re-recruited healthy volunteers and performed MHC class I and\nMHC class II matching with the nasopharyngeal carcinoma cells used in this study (it\nis nearly impossible to find volunteers whose MHC class I and MHC class II molecules\nfully match the nasopharyngeal carcinoma cell line). We then sorted CD8+ T cells from\nMHC class I-matched volunteers and CD4+ T cells from MHC class II-matched\nvolunteers, which were subsequently co-cultured with the nasopharyngeal carcinoma\ncells. We found that overexpression of BALF2-HR in nasopharyngeal carcinoma cells,\nwhich upregulates MHC class II expression, does moderately activates CD4+ T cells\n(Fig. S4E). However, the cytotoxic effect of isolated CD4+ T cells on tumor cells was\nlimited (Fig. S4F). This is consistent with the role of CD4+ T cells in anti-tumor\nimmunity, primarily supporting CD8+ T cells. Furthermore, co-cultured CD8+ T cells\nwere significantly induced to undergo apoptosis and exhaustion (Fig. 2C-G). We sorted\nisolated CD8+ T cells and CD4+ T cells, repeated the experiments, and replaced the\ntumor cell and T cell co-culture experiments in this study. We believe these revised\nexperiments can prevent immune rejection caused by HLA mismatching, making the\nresults more reliable. In summary, we found that BALF2-HR significantly inhibits the\nkilling ability of CD8+ T cells and promotes their exhaustion. This study primarily\nfocuses on its regulatory effect on CD8+ T cells. Additionally, BALF2-HR also exhibits\ncertain activating effects on CD4+ T cells, which require further investigation.\nAdditionally, based on your suggestion, we have added the results related to the\nTCR signaling pathway in CD4+ T cells with and without anti-LAG3 monoclonal\nantibody treatment (Fig. S10I). The results indicate that BALF2-HR, by upregulating\nMHC class II molecules, can partly activate CD4+ T cells. When a LAG3 monoclonal\nantibody is added, the activation of CD4+ T cells becomes more evident.\nFurthermore, as the authors discussed in response to my query #6, since “the\nimmunosuppressive function of LAG-3 is not solely dependent on HLA-II molecules”,\nthis new mechanism of LAG3 immunosuppression is one addition to the previous\nfindings. This needs to be clarified in the discussion section.\nResponse：Thank you for this insightful suggestion. We have now clarified this point\nin the revised Discussion section to better explain our findings within the broader\ncontext of LAG-3 biology.\n2. The variation in the EBV genome in NPC cells attributed to latency type II is\nplausible. However, extranodal NK/T cell lymphoma and Hodgkin lymphoma are also\nlatency type II malignancies (PMID: 26449716). Therefore, the authors' explanation is\ninsufficient to account for the variation in the EBV genome in NPC cells.\nResponse：The variation of the EBV genome in nasopharyngeal carcinoma (NPC) may\nresult from the complex interactions between the virus and host cells, involving immune\nevasion mechanisms, host genetic susceptibility, viral replication strategies, and the\ntumor microenvironment. Host immune evasion, HLA gene diversity, and the latent\nstate of the virus all contribute to the variation of the EBV genome. Immune\nsuppression and chronic inflammation in the tumor microenvironment promote long-\nterm viral latency, which raises the risk of mutations. Additionally, host cell genome\nvariations may affect immune responses and viral clearance, enabling the virus to\npersist and spread regionally by adapting to the nasopharyngeal epithelial\nmicroenvironment.\n3. Regarding the clustering analysis in Figure S1F, information on the marker genes\nused to identify each cluster and their expression levels should be provided.\nResponse：Thank you for your insightful comment. We understand the importance of\nproviding detailed information on the marker genes used in the clustering analysis\nshown in Figure S1F. To address this, we have added Figure S1G, which lists the marker\ngenes for each cluster along with their corresponding expression levels.\nReviewer #3 (Remarks to the Author):\nI have reviewed the authors’ responses to my comments. All of my questions and\nconcerns have been addressed appropriately. The missing references I requested have\nalso been added.\nI have no further comments at this stage.\nResponse：Thank you very much!\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse：Thank you very much!"}, {"round": 3, "reviewer_comments": {"reviewer_2": "In this manuscript by Ge J. et al., the authors faithfully responded to my concerns. Yet, some points are not clear and need to be clarified.\n1. The authors’ new data have addressed my point. However, some sectiions, such as lines 436-437 and 647-650, require a more detailed description based on the new data. For example, Fig. S10I shows that MHC-II-LAG3 interactions were not a significant mechanism of CD4+ T cell suppression, though anti-LAG3 mAb enhanced ZAP70 signaling. Therefore, other suppressive mechanisms through LAG3 may act on CD4+ T cells.\n2. While I understand the difficulty of explaining the importance of BALF2-HR only in NPC cells, the authors' argument is unclear. They should more directly explain why this variation is involved in NPC development but not in other EBV-associated malignancies by referencing previous reports and their own data."}, "author_response": "Response to the reviewer\nReviewer #2 (Remarks to the Author):\nIn this manuscript by Ge J. et al., the authors faithfully responded to my concerns. Yet, some points\nare not clear and need to be clarified.\n1. The authors’ new data have addressed my point. However, some sections, such as lines 436-437\nand 647-650, require a more detailed description based on the new data. For example, Fig. S10I\nshows that MHC-II-LAG3 interactions were not a significant mechanism of CD4+ T cell\nsuppression, though anti-LAG3 mAb enhanced ZAP70 signaling. Therefore, other suppressive\nmechanisms through LAG3 may act on CD4+ T cells.\nResponse: Thank you for your thoughtful comment. We appreciate your recognition that the new\ndata have addressed your previous concerns. In response to your suggestion, we have now revised\nthe sections in lines 436-437 and 647-650 to provide a more detailed description based on the\nupdated data.\nWe hope this revision clarifies the mechanism and provides a more comprehensive understanding\nof the role of LAG3 in CD4+ T cell suppression. Thank you again for your valuable feedback.\n2. While I understand the difficulty of explaining the importance of BALF2-HR only in NPC cells,\nthe authors' argument is unclear. They should more directly explain why this variation is involved\nin NPC development but not in other EBV-associated malignancies by referencing previous reports\nand their own data.\nResponse: Thank you for your insightful feedback. We appreciate your recognition of the\ncomplexity involved in explaining the specific role of BALF2-HR in NPC cells, and we concur that\na clearer and more direct explanation is necessary. In response to your suggestion, we have revised\nthe Discussion sections to provide a more detailed and cohesive explanation of why the BALF2-HR\nvariant is implicated in NPC development but not in other EBV-associated malignancies.\nIn nasopharyngeal carcinoma (NPC), EBV exhibits a distinct \"abortive lytic\" state, in which both\nlatent genes (e.g., EBNA1, LMP1/2) and early lytic genes, such as BALF2, are expressed\nabnormally and simultaneously[1-3]. This dual-expression pattern is rare in other EBV-related\nmalignancies, such as Burkitt lymphoma and Hodgkin lymphoma, and it likely forms the molecular\nbasis for the specific involvement of BALF2 in NPC pathogenesis. Furthermore, populations in\nhigh-incidence regions of NPC carry specific HLA mutations, such as SNPrs2860580 and\nrs2894207, which have been shown to interact synergistically with BALF2 gene variants,\nparticularly coding region amino acid polymorphisms[4]. This interaction could improve the\nefficiency of viral peptide presentation, change how the immune system recognizes BALF2, and\ncollectively enhance the risk of malignant transformation in nasopharyngeal epithelial cells.\nImportantly, this synergy is highly specific to NPC, as it is not observed in other EBV-associated\ncancers.\nAdditionally, environmental exposures in high-incidence regions, such as consuming nitrite-rich\nfoods, may further exacerbate the carcinogenic effects of BALF2[5]. Nitrite metabolites might\nactivate BALF2 transcription, and the accumulation of reactive oxygen species (ROS) could\naccelerate EBV genomic mutations, strengthening the virus's oncogenic potential. This\n\"environment-virus\" interaction is much weaker in low-risk areas, further supporting the regional\nspecificity of BALF2's role in NPC. Phylogenetic analysis of EBV strains from endemic regions\nalso reveals significant positive selection signals in the BALF2 gene, with mutation patterns\ncorrelating with geographical distribution [6]. This region-specific evolution may allow EBV to\nbetter evade immune surveillance and induce cellular transformation in certain populations,\nreinforcing the connection between BALF2 and NPC endemicity.\nIn conclusion, BALF2’s involvement in NPC pathogenesis is driven by its complex interactions\nwith the host genetic background, environmental exposures, and viral evolution. We believe that the\nrevised discussion section now provides a clearer and more comprehensive explanation of the\nmechanisms linking BALF2 to NPC development in endemic regions.\nReferences\n1. Jochum, S., et al., RNAs in Epstein-Barr virions control early steps of infection. Proc Natl\nAcad Sci U S A, 2012. 109(21): p. E1396-404.\n2. Xu, X., et al., EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression\nthrough recruiting monocytes and regulating their directed differentiation. PLoS Pathog, 2024.\n20(1): p. e1011934.\n3. Arvey, A., et al., An atlas of the Epstein-Barr virus transcriptome and epigenome reveals host-\nvirus regulatory interactions. Cell Host Microbe, 2012. 12(2): p. 233-45.\n4. Xu, M., et al., Host genetic variants, Epstein-Barr virus subtypes, and the risk of\nnasopharyngeal carcinoma: Assessment of interaction and mediation. Cell Genom, 2024. 4(2): p.\n100474.\n5. Ward, M.H., et al., Dietary exposure to nitrite and nitrosamines and risk of nasopharyngeal\ncarcinoma in Taiwan. Int J Cancer, 2000. 86(5): p. 603-9.\n6. Xu, M., et al., Genome sequencing analysis identifies Epstein-Barr virus subtypes associated\nwith high risk of nasopharyngeal carcinoma. Nat Genet, 2019. 51(7): p. 1131-1136."}, {"round": 4, "reviewer_comments": {"reviewer_2": "The authors have adequately addressed my concerns. Therefore, I recommend accepting the manuscript for publication."}, "author_response": ""}]

Decision letter