Cancer vaccine overcomes immune evasion of nasopharyngeal carcinoma by restoring MHC-I through transcriptional regulation of NLRC5

Cancer vaccine overcomes immune evasion of nasopharyngeal carcinoma by restoring MHC-I through transcriptional regulation of NLRC5 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cancer vaccine overcomes immune evasion of nasopharyngeal carcinoma by restoring MHC-I through transcriptional regulation of NLRC5 Chai Phei Gan, Sau Yee Kok, Bernard Kok Bang Lee, Natasha Zulaziz, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7037136/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Nasopharyngeal carcinoma (NPC) is considered an immune-hot tumour. However, 30–80% of cases exhibit downregulation of antigen processing and presentation machinery (APM), enabling the evasion of host immunosurveillance. While cancer vaccines aim to trigger immune responses against tumour antigens, their efficacy in APM-deficient tumours remains uncertain. This study evaluates the efficacy of cancer vaccine targeting tumour-associated antigens in APM-downregulated NPC cells in vitro and further validates the modulation of APM pathways in vivo . Methods APM status was evaluated via differential gene expression analysis of 42 NPC tumours and 4 non-NPC tissues using a 17-gene APM signature. Genes downregulated in NPC and associated with reduced MHC-I expression were identified. MHC-I expression was further examined by immunohistochemistry in 35 tumours and 5 non-NPC tissues. The effect of cancer vaccine on APM gene expression was examined by co-culturing peptide-trained T cells with NPC cells. Next, T cell-mediated cytotoxicity was assessed in an APM-deficient model generated by siRNA-mediated knockdown of NLRC5 . Finally, cancer vaccine-induced modulation of APM genes was validated in a poorly immunogenic mouse tumour model. Results Among the 17-gene APM signature, NLRC5 was the most significantly downregulated gene in NPC and strongly correlated with reduced MHC-I expression. Immunohistochemistry confirmed MHC-I downregulation in 63% of tumours. Co-culture of NPC cells with peptide-trained T cells upregulated NLRC5, and key MHC-I assembly genes (TAP1 and B2M), enhancing MHC-I expression and antigen-specific cytotoxicity in NPC cells expressing the target antigen. Notably, the knockdown of NLRC5 was reversed upon co-culture with peptide-trained T cells, resulting in T cell-mediated cytotoxicity. In vivo , cancer vaccine treatment consistently induced APM gene expression, including NLRC5, supporting its potential in restoring antigen presentation in NPC. Conclusions This study demonstrates that peptide-trained T cells can upregulate NLRC5 and MHC-I expressions on tumour cells, thereby restoring antigen presentation and enhancing tumour immunogenicity. These findings underscore the therapeutic potential of cancer vaccines in treating APM-downregulated NPC. Nasopharyngeal Cancer Cancer Vaccine Tumour Associated Antigen Antigen Presentation Machinery Immunogenicity T Cell Cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Nasopharyngeal carcinoma (NPC) is a subset of head and neck cancer, and exhibits a distinct population-specific prevalence, occurring nearly 40 times more frequently in Asians than in Caucasians ( 1 ). Due to its non-specific early symptoms, over 80% of patients are diagnosed at a locally advanced stage or with distant metastasis ( 2 ). While early and locally advanced NPC respond well to radiotherapy, recurrent or metastatic NPC carries a poor prognosis, with a median overall survival of 20 months ( 3 ). Immune checkpoint inhibitors (ICIs) are increasingly used to treat patients with recurrent or metastatic disease, considering that NPC is an immune hot tumour characterized by its association with Epstein-Barr virus (EBV) infection, dense lymphocytic infiltration and elevated PD-L1 expression ( 4 ). However, overall response rates to anti–PD-1 monotherapy remain modest at 10–43% and fail to produce a durable clinical benefit ( 5 , 6 ). One proposed reason for this limited efficacy is the deficiency in antigen processing and presentation machinery (APM), which is essential for effective T cell-mediated anti-tumour immunity ( 7 – 9 ). Downregulation of the APM pathway has been reported in 30–80% of NPC patients ( 10 – 13 ). Specifically, the expression of major histocompatibility complex (MHC-I) molecules, including the HLA-A and HLA-B, as well as their associated components such as TAP1 and TAP2, is significantly reduced in NPC compared to normal tissue. APM downregulation can result from “hard/irreversible” genomic alterations, such as chromosomal structural variants, single-nucleotide variants and mutations, which lead to inactivation of HLA-A and HLA-B in 13% and 9% of NPC, respectively ( 10 , 14 ). “Soft/reversible” alterations driven by epigenetic, transcriptional and posttranscriptional mechanisms may also contribute to APM downregulation, although their prevalence in NPC remains unclear ( 15 , 16 ). Additionally, EBV-encoded proteins such as BNLF2a and BILF1 can also cause APM downregulation ( 10 , 17 ). MHC-I downregulation is associated with poorer survival outcome, likely due to compromised antigen presentation, enabled cancer cells to evade immune detection ( 11 , 14 , 18 ). Recent research has demonstrated the potential of targeting APM to overcome immune escape in cancer by promoting tumour antigenicity, enhancing immunogenicity, and stimulating T cell-mediated tumour killing. For example, platinum-based chemotherapies have been shown to upregulate MHC-I on both tumours and dendritic cells in preclinical studies, thereby improving the efficacy of anti-PD1 thereapy ( 19 ). In NPC, combining anti-PD1 therapy with gemcitabine and cisplatin has led to improved response rates of 70–91% ( 6 , 20 ), likely due to chemotherapy-induced immune modulation. This effect may be mediated by increased MHC-I antigen presentation, potentially through activation of pathways such as IFNβ and NF-κB ( 21 ). However, despite these immunostimulatory effects, the combination of chemotherapy and anti-PD1 is associated with substantial toxicity, with grade ≥ 3 adverse events reported in 57–94% of NPC patients. These limitations underscore the need for alternative strategies that can enhance antigen presentation with fewer side effects. Cancer vaccines targeting tumour-associated antigens (TAAs) offer a promising approach to provoke an immune response by priming tumour-reactive T cells against existing tumours ( 22 , 23 ). Notably, MAGED4B and FJX1 are highly immunogenic TAAs that are overexpressed in NPC ( 24 , 25 ). Vaccines targeting these TAAs significantly inhibited tumour growth when combined with anti-PD1 therapy ( 26 ). In this study, we investigated the potential of cancer vaccines targeting MAGED4B and FJX1 in modulating APM to restore anti-tumour immunity in NPC. Specifically, we demonstrated that co-culturing peptide-trained T cells and NPC cells endogenously expressing these TAAs enhances antigen presentation through the upregulation of NOD-like receptor family CARD domain containing 5 (NLRC5) and MHC-I in the APM pathway to induce T cell-mediated cytotoxicity. Material and methods Bioinformatics analyses of APM gene expression The RNA sequencing dataset GSE68799, comprising 41 NPC and 4 non-NPC tissues, was obtained from the Gene Expression Omnibus database. This dataset was selected for its large number of tumour samples and its accessibility through a public repository. A curated APM signature consisting of 17 genes ( 27 ) was used to identify differentially expressed genes (DEGs) between NPC and non-NPC tissues using DESeq2, with thresholds set at |log2 fold change| >1 and p -value < 0.05 ( 28 ). A heatmap illustrating gene expression patterns was generated using the Morpheus web tool ( https://software.broadinstitute.org/morpheus ). APM genes that were significantly downregulated in NPC tissues were further analysed using Pearson correlation to assess their association with MHC-I genes (HLA-A, HLA-B, and HLA-C). Additionally, the KEGG antigen processing and presentation pathway was analysed in the GSE68799 dataset to assess the prevalence of HLA gene downregulation in NPC. Immunohistochemistry staining Formalin-fixed, paraffin-embedded (FFPE) tissues were used to assess target protein expression via immunohistochemistry (IHC) staining with the EnVision + system (Dako, Germany). These included whole tissue sections from tumours ( n = 14) and a tissue microarray (NH1001a; TissueArrays.com, USA) comprising tumour samples ( n = 21) and non-NPC tissues ( n = 5). The whole tissue sections were obtained from Penang General Hospital upon medical ethics approval (NMRR-09-944-4848). FFPE tissue sections were deparaffinized and dehydrated in decreasing grades of ethanol. Antigen retrieval was performed by boiling the tissues in the respective antigen retrieval buffer in the microwave. The Tris/EDTA pH 9.0 buffer was used for antigen retrieval of FJX1, while sodium citrate pH 6.0 buffer was used for antigen retrieval of MHC-I, MHC-II and MAGED4B. Tissues were incubated with 1% bovine serum albumin in Tris-buffered saline to prevent unspecific binding. The primary antibodies were anti-HLA Class-I monoclonal antibody, clone EMR8-5 (1:100; Abcam, USA), anti-HLA Class-II monoclonal antibody clone 6C6 (1:100; Abcam, USA), anti-MAGED4B (1:100; Sigma-Aldrich, USA) and anti-FJX1 (1: 200; Sigma-Aldrich, USA). After peroxidase blocking, antigen expressions were visualized using the Envision + Dual Link System HRP (DAB+) kit (Dako, Germany). The immunoreactivity of MHC-I was evaluated based on the criterion established by the Human Leukocyte Antigen and Cancer component of the 12th International Histocompatibility Workshop ( 29 ). The staining intensity of MHC-I in the lymphocytes and vessel endothelium is regarded as internal positive controls. Tumour cells stained as similar or greater intensity than the internal positive control was graded, and area positivity score was given as 0 (no expression): less than 25%; 1 (low expression): 25 to 75% and 2 (high expression): greater than 75%. On the other hand, the immunoreactivity of MAGED4B and FJX1 was scored based on a 4-point intensity scoring system: 0 = negative expression; 1 = weak positive; 2 = moderate positive; 3 = strong positive. Score 0 and 1 were grouped as low expression, 2 and 3 were grouped as high expression, as reported previously ( 25 ). Cell culture and cell transfection NPC-43 and NPC-53 cells, derived from recurrent tumours ( 30 ), and NPC-268 cell derived from a primary tumour ( 31 ) were cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), 100 IU Penicillin/Streptomycin (Gibco, USA) and 4 µM Rock inhibitor (Y-27632; Enzo Life Sciences, USA). Cells were maintained at 37°C in 5% CO 2 incubator. NPC cell lines used in this study were authenticated by short tandem repeat DNA profiling to confirm their identity and rule out cross-contamination. Additionally, they were tested and confirmed to be mycoplasma-free. To knockdown NLRC5 expression, NLRC5 siRNA (5’ GGA CAC CUG GCA GUC UUU CAU UCA U3’; Invitrogen, USA) was transfected into NPC cells using Lipofectamine™ RNAiMAX (Invitrogen, USA) according to the manufacturer’s protocol. For control, cells were transfected with non-targeting (NT) siRNA (Invitrogen, USA). To evaluate the transfection efficiency of NLRC5 knockdown, cells were harvested at the indicated time points for real-time PCR and western blot assays. The mouse melanoma cell line B16F10 expressing the human-HLA-A2 gene (B16F10-A2) was cultured in RPMI-1640 medium (Gibco, USA) containing 10% FBS and 100 IU Penicillin/Streptomycin (Gibco, USA), at 37°C in an incubator with 5% CO 2 . To express MAGED4B and FJX1 in B16F10-A2 cells, the pLENTI-6.3 plasmids carrying MAGED4B and FJX1 were simultaneously transfected into the cells using Lipofectamine according to manufacturer’s instruction. Cells were further selected using antibiotics (G418 and blasticidin) for one month. Western blotting was performed to confirm the expression of MAGED4B and FJX1 in B16F10-A2 prior to their use in the mouse experiment. Generation of MAGED4B- and FJX1-specific T cells Peripheral blood mononuclear cells (PBMCs) were harvested from blood samples collected into cellular preparation tubes (Becton Dickinson, USA) from three healthy donors (HD30, HD31 and HD32). Red blood cells were removed using Ammonium Chloride Potassium (Gibco, USA) lysing buffer. The use of peripheral blood samples in this study was approved by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [DF OS 2101/0002/2179(L). CD8 + T cells were isolated through negative selection using the human CD8 + T cell isolation kit (Miltenyi Biotec, Germany). Both CD8 + and non-CD8 + cell fractions were collected, with the non-CD8 + fraction serving as autologous antigen-presenting cells (APC). The CD8 + T cells were cultured in RPMI medium supplemented with 10% FBS and 30ng/ml anti-CD3 (OKT3 clone, Miltenyi Biotec, Germany). A ntigen-specific T cells were generated using a modified protocol adapted from Erskine et al. 2012 ( 21 ). CD8 + T cells were stimulated with 10µg/mL of overlapping peptide pools derived from MAGED4B (M-OPP) and FJX1 (F-OPP; JPT Peptide Technologies, Germany), respectively. CD8 + T cells were supplemented with 50 IU/mL of recombinant human interleukin-2 (IL-2; R&D Systems, USA) from day 3 onwards. Restimulation with autologous APC cells was performed at day 7 and 14. To prepare APCs, the non-CD8 + fraction was pulsed with 10 µg/mL of M-OPP or F-OPP, followed by mitomycin C treatment to inhibit cell proliferation. CD8⁺ T cells that completed 3 rounds of stimulation were hereafter referred to as peptide-trained T cells and were used in subsequent functional assays. Modulation of APM gene expression by peptide-trained T cells The expression of APM genes in NPC cell lines following exposure to peptide-trained T cells was assessed in vitro . NPC cells (target) were seeded overnight at 3 × 10 5 in a 6-well plate, then co-cultured with peptide-trained T cells (effector) at an effector-to-target ratio of 1: 10 for 24 hours. NPC cells stimulated with 100 ng/mL IFNγ (R&D Systems, USA) served as a positive control. Following incubation, total RNA was extracted from NPC cell lines using RNeasy Micro kit (Qiagen, Germany), and cDNA was synthesized from 1µg of total RNA using BIOLINE SensiFAST reagent kit (Bioline Reagent Ltd, UK). Gene expression analysis was performed on the 7500 Real-Time PCR System (Applied Biosystems, USA) using specific PCR primers (Supplementary Table 1) and Power Sybr Green PCR Master Mix (Applied Biosystems, USA). Relative mRNA levels of NLRC5, CD274, TAP1 , and B2M were obtained after normalisation to GAPDH . Flow cytometry analysis The expression of human leukocyte antigen ABC (HLA-ABC), corresponding to MHC-I was analysed by flow cytometry. Firstly, HLA-A typing was performed on PBMCs and NPC cell lines using specific antibodies and isotype controls. Briefly, cells were stained with FVS780 viability dye (BD Biosciences, USA) for 15 minutes followed by incubation with the respective primary antibodies: PE-conjugated mouse anti-human HLA-A2 (Clone BB7.2, BD Biosciences, USA), unconjugated HLA-A11 +/-A3 IgG (One Lambda, USA), unconjugated HLA-A2 and A24 IgM (One Lambda, USA), and unconjugated HLA-A24, A11, A2403 + IgM (One Lambda, USA). For isotype controls, cells were stained with mouse IgG2b-UNLB (SouthernBiotech, USA) and mouse IgM-UNLB (SouthernBiotech, USA). Cells stained with PE-conjugated mouse anti-human HLA-ABC (Clone W6/32, BD Biosciences, USA, 567582) served as the positive control. Unconjugated primary antibodies and isotype controls were further incubated with the corresponding secondary antibodies: Goat F(ab')₂ anti-mouse IgG (H + L), human ads-PE (SouthernBiotech, USA), and Goat F(ab')₂ anti-mouse IgM, human ads-PE (SouthernBiotech, USA). After staining, a minimum of 1 × 10 5 cells were analysed using the LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, USA), with gating against unstained and isotype controls. To assess total MHC-I expression on NPC cells, cells were stained with FVS780, followed by HLA-ABC staining, and analysed as described above. The median fluorescence intensity (MFI) of HLA-ABC was quantified using FlowJo software (BD Biosciences, USA) to determine the relative abundance of HLA-ABC among the NPC cell lines. Western blot analysis NPC cell lines were lysed in RIPA buffer (Thermo Scientific, USA) supplemented with 1x Halt protease and phosphatase inhibitors cocktail (Thermo Scientific, USA). Protein concentrations were measured using Pierce BCA protein assay kit (Thermo Scientific, USA). Protein lysates were denatured by heating at 98°C for 10 minutes. Equal amounts of protein were separated on a 10–12% SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk and probed with antibodies against MAGED4B (1:1000, HPA003554, Sigma-Aldrich, USA), FJX1 (1:1000, HPA059220, Sigma-Aldrich, USA), HLA Class I A/B/C (1:1000, clone EMR8-5, Ab70328, Abcam, USA), and NLRC5 (1:500, clone 3H8, MABF260, Millipore, USA). Anti-GAPDH antibody (1:1000, Proteintech, USA) and anti-tubulin (1:1000, T5168, Sigma-Aldrich, USA) were used as a loading control. An Immobilon Forte Western HRP substrate (Milipore, USA) was used for chemiluminescence detection and images taken using Western blot imager (Azure Biosystem, USA). Suberoylanilide hydroxamic acid (SAHA) treatment assay NPC cells were seeded at 3x10 5 cells per well in a 6-well plate overnight and serum-starved for 24 hours. Cells were treated with SAHA (Cayman Chemical, USA) at a final concentration of 1 µM, 5 µM and 10 µM for 24 hours in the CO 2 incubator. Subsequently, cells were harvested to assess MHC-I protein expression using HLA-ABC MFI quantification by flow cytometry and Western blotting. T cell-mediated cytotoxicity assay The cytotoxic activity of peptide-trained T cells against NPC cell lines was assessed using the xCELLigence Real-Time Cell Analyzer (RTCA; Agilent, USA). Briefly, background impedance values of 96 well E-Plate (Agilent, USA) filled with medium were obtained. NPC target cells were seeded into the E-plate (4x10 4 cells/well for NPC43 and NPC53 or 2x10 4 cells/well for NPC268), and allowed to adhere for 24 hours in the RTCA. peptide-trained T cells were added to target cells at a 10:1 effector-to-target ratio. Impedance values were recorded hourly for 24 hours. Cell index was normalized to the time of effector T cells addition, and cytolysis was calculated in relative to control cells without effector T cells. Animal experiment AAD mice (transgenic for HLA-A2.1/H2-Dd allele) were bred and housed in the Animal Facility Centre of National University Malaysia. AAD mice (6–10 weeks) were inoculated with 1 x10 5 B16F10-A2 cell line expressing MAGED4B/FJX1 and were randomised into 2 groups to receive (i) 40µg plasmid DOM (pLDOM, control); (ii) 40 µg each of doggybone DNA vaccine encoding MAGED4B/FJX1 (DNA vaccine); intramuscular aided by electroporation administration of DNA vaccine was given on day 5 and day 21 post cell inoculation; Tumour size was monitored 2 times/week until day 40. Tumour volumes were evaluated using the formula: volume = ½ (length X width 2 ). Animal study was approved by Experimental Animal Ethics Committee, National University Malaysia (CRM/2020/KUE PENG/25-MAR./1093-APR.-2020-APR.-2023). RNA sequencing and bioinformatic analysis Total RNA was extracted from mouse tumour tissue using the Qiagen RNeasy mini kit (Qiagen, Germany), with purity and integrity assessed via Nanodrop (Thermo Scientific, USA) and the RNA Nano 6000 Assay Kit run on the Bioanalyzer 2100 system (Agilent, USA). RNA library was prepared from 1 µg of total RNA using the NEBNext® UltraTM RNA Library Prep Kit (NEB, USA) and sequenced on Illumina NovaSeq (Illumina, USA) to generate paired-end 150bp reads. Raw reads (FASTQ format) were processed with fastp to remove adapter sequences, poly-N regions, and low-quality reads. Clean reads were mapped to the reference genome using HISAT2. DEGs between the DNA vaccine and control groups were identified using a joint filtering method, with thresholds for up-regulated and down-regulated genes set at |log 2 fold change| >1 and p -value < 0.01 ( 18 ). To further analyse the functional significance of these DEGs, the KEGG pathway enrichment analysis was performed using Metascape (Broad Institute, USA) ( 22 ). A heatmap of genes in the 17-gene APM signature was generated to examine the changes in gene expression between the DNA vaccine and control group. Statistical analysis Statistical analyses for bioinformatics studies are described in the respective sections. Experimental data were analysed using GraphPad Prism 10 (GraphPad Software, USA). IHC scoring of MHC-I expression in NPC tissues was evaluated using Fisher’s exact test to determine associations with clinical parameters. The expression of APM genes in NPC cells stimulated by peptide-trained T cells or IFNγ was compared to control cells using one-way ANOVA followed by Bonferroni’s post-hoc test for multiple comparisons. NLRC5 expression after siRNA knockdown, antigen-specific T cell-mediated killing, tumour growth in animal models, and histopathological assessment of immune cell infiltration were analysed using Student’s t-test. Statistical significance was set at p < 0.05 for all analyses. All data were reproduced with at least two independent experiments. Results Downregulation of NLRC5 expression is associated with decreased MHC-I expression in NPC tumours Gene expression analysis of the NPC dataset (GSE68799) revealed that genes within the APM signature, as defined by Thompson and colleagues ( 27 ), were collectively expressed at lower levels in NPC tumours compared to non-NPC tissues (Fig. 1 a). Among the 17 APM genes, NLRC5, CIITA, PSMB10, PSME1 and PSMB9 were significantly downregulated in NPC. Specifically, NLRC5 showed the most pronounced reduction at 2.2-fold. Given that NLRC5 functions as a master transcriptional regulator of MHC class I genes, its expression showed a strong positive correlation with the expression of HLA-A, HLA-B , and HLA-C . (Fig. 1 b). In comparison, PSMB10 and PSMB9 exhibited moderate positive correlation, while CIITA and PSME1 showed only weak correlation with the expression of HLA-A, HLA-B , and HLA-C (sFig. 1a). Tumours with low NLRC5 expression consistently exhibited low HLAs expression, indicating that NLRC5 downregulation may lead to impaired transcriptional activation of the MHC-I group of genes, potentially decreasing MHC-I expression in NPC. Furthermore, analysis of the KEGG antigen processing and presentation pathway in the GSE68799 dataset revealed that approximately 60–70% of NPC cases exhibit downregulation of HLA s (sFig. 1b). MHC-I downregulated tumours overexpress MAGED4B and FJX1 To validate the DEG findings indicating MHC-I downregulation, we assessed MHC-I and MHC-II protein levels by IHC in an independent cohort of 35 NPC and 5 non-NPC tissues. The demographic and clinical characteristics of NPC patients are shown in Table 1 . In non-NPC tissues, MHC-I expression was observed in normal epithelial cells at levels that are comparable or higher than those in the surrounding stroma. By contrast, NPC tumour cells exhibited weaker MHC-I membrane staining compared to lymphocytes and endothelial cells in the surrounding stroma, indicating MHC-I downregulation in tumours (Fig. 2 a). MHC-I downregulation was detected in 63% (22/35) of NPC cases, and none in the non-NPC tissues (Fig. 2 b). The IHC findings are consistent with our gene expression analysis, confirming that MHC-I is significantly downregulated in about two-thirds of NPC cases. However, MHC-I downregulation was not associated with disease stage or lymph nodes involvement (Table 2 ). Meanwhile, MHC-II downregulation showed minimal differences between NPC and non-NPC tissues (29% vs 40%). Given the higher prevalence of MHC-I downregulation in NPC, further investigation into its expression and modulation was conducted in this study. To evaluate the relationship between TAAs expression and MHC-I status, the same set of tissues underwent IHC staining for MAGED4B and FJX1. Both TAAs were found to be overexpressed in 91% of NPC tissues (Table 3 a; sFig. 2a), which is in line with previous study ( 25 ). However, about 60% of NPC cases with high TAAs expression exhibited MHC-I downregulation, indicating a need for strategies to restore antigen presentation to enable effective immune surveillance and promote antigen-specific cytotoxicity (Table 3 b). Table 1 Demographic and clinical characteristics of NPC patients. Variables Patients with NPC, N (%) NPC tissues 35 (100%) Whole tissue section 14 (34.1%) TMA 21 (65.9%) Age Range, years 28–84 Mean ± S.D. 51.2 ± 14.5 Gender Male 32 (91.4%) Female 3 (8.6%) Staging Stage I-II 22 (62.9) Stage III-IV 11 (31.4) Lymph nodes metastasis Negative 20 (57.1) Positive 13 (37.1) Table 2 The association between MHC-I and MHC-II with clinical variables. Variables MHC-I MHC-II Low High p -value Low High p -value Sample type NPC tumours 22 (62.9) 13 (37.1) 0.0289* 10 (28.6) 25 (71.4) 0.6266 Non-NPC 0 (0.0) 4 (100.0) 2 (40.0) 3 (60.0) Staging Stage I-II 14 (40.0) 8 (22.9) 0.7136 5 (14.3) 17 (48.6) 0.4376 Stage III-IV 6 (17.1) 5 (14.3) 4 (11.4) 7 (20.0) Lymph nodes metastasis Negative 13 (37.1) 7 (20.0) 0.7171 3 (8.6) 17 (48.6) 0.1067 Positive 7 (20.0) 6 (17.1) 6 (17.1) 7 (20.0) The association between MHC-I and MHC-II expression with clinical variables was examined with the Fisher’s exact test. *A p -value < 0.05 is considered to be statistically significant. Table 3 a : The association between target antigens expression in NPC tumours and non-NPC tissues. Variables MAGED4B FJX1 Low High p -value Low High p -value Sample type NPC tumours 3 (8.6) 32 (91.4) < 0.0001* 3 (8.8) 31 (91.2) 0.0005* Non-NPC 5 (100.0) 0 (0.0) 4 (100.0) 0 (0.0) The association between MAGED4B and FJX1 expression with sample type was examined with the Fisher’s exact test. *A p -value < 0.05 is considered to be statistically significant. Table 3 b : TAAs expression and MHC-I status in NPC tissues. Protein expression MAGED4B (n = 35) FJX1 (n = 34) Low High Low High MHC-I Low 1 (2.9%) 21 (60%) 2 (5.9%) 20 (58.8%) High 2 (5.7%) 11 (31.4%) 1 (2.9%) 11 (32.4%) Peptide-trained T cells enhance MHC-I expression in NPC cells To investigate the effect of peptide-trained T cells on MHC-I expression in NPC cells, we first screened a panel of NPC cell lines for baseline expression of MAGED4B, FJX1 and MHC-I. Both MAGED4B and FJX1 were detected across all NPC cell lines (sFig. 3a). Flow cytometry analysis revealed that NPC-268 exhibited the lowest surface MHC-I expression, followed by NPC-43, and NPC-53 (Fig. 3 a). All cell lines showed MHC-I induction upon IFNg stimulation. CD8 + T cells specific for MAGED4B (M-T cells,) and FJX1 (F-T cells) were generated from healthy donors (HD23, HD30 and HD32) carrying at least one of these HLA haplotypes: HLA-A2, HLA-A11 or HLA-A24 (Table S3 ). These peptide-trained T cells were co-cultured with HLA-A matched NPC cell lines (Table S2 ), resulting in an approximately two-fold increase in MHC-I expression in NPC cells relative to baseline levels (Fig. 3 b). Epigenetic modulation such as DNA methylation and histone deacetylation (HDAC) is known to contribute to MHC-I downregulation in tumours. As the NPC cell lines used in this study were hypomethylated, DNA methylation was considered an unlikely mechanism of MHC-I suppression ( 31 , 32 ). Although histone deacetylation status of these cell lines was not fully characterized, HDAC inhibition was performed as an indirect approach to probe HDAC regulation of MHC-I in these cell lines. SAHA was chosen as the HDAC inhibitor in this study because it is currently being tested in combination with ICI for recurrent and metastatic head and neck cancer ( 33 ). The response to SAHA treatment varied across NPC cell lines. NPC-43 exhibited a dose-dependent increase in total and surface MHC-I expression, indicating the potential of HDAC in mediating MHC-I expression in this cell. Meanwhile, NPC-53 and NPC-268 showed minimal changes in surface MHC-I, with relative fold change below 2 (Fig. 3 c). In comparison to SAHA treatment, peptide-trained T cells induced MHC-I upregulation across all three NPC cell lines, suggesting that a vaccine-based approach may offer a more robust strategy for enhancing MHC-I expression in these NPC cell lines. Peptide-trained T cells upregulate MHC-I-associated genes and promote antigen-specific killing of NPC cell lines To elucidate the molecular basis of MHC-I upregulation in NPC by peptide-trained T cells, we analysed the expression of key genes involved in MHC-I transcription and assembly including NLRC5 , TAP1 , and B2M following co-culture. Given that IFNγ stimulation is known to concurrently induce both MHC-I and CD274 (PD-L1), which can have opposing effects on antitumor immunity ( 34 ), we also examined CD274 expression in response to peptide-trained T cells. Compared to control cells, NLRC5, TAP1 and B2M mRNA levels were significantly upregulated in NPC cells following co-culture with peptide-trained T cells, although the magnitude of induction varied across cell lines (Fig. 4 a). Notably, CD274 expression was substantially lower following co-culture with peptide-trained T cells compared to direct IFNγ stimulation. These findings indicate that peptide-trained T cells preferentially enhance MHC-I while limiting excessive PD-L1 upregulation, which may help to overcome immune suppression. Importantly, antigen-specific T cells killing was observed across all three NPC cell lines following MHC-I upregulation induced by peptide-trained T cells (Fig. 4 b). After 24 hours of co-culture, these peptide-trained T cells achieved cytolytic efficiencies ranging from 50–60%, exceeding the 20% baseline for non-specific killing, regardless of the differences in basal MHC-I expression in these cell lines. For example, NPC-268, which initially exhibited low MHC-I levels, showed enhanced MHC-I expression and demonstrated susceptibility to T cell-mediated cytotoxicity, achieving a similar cytolysis rate compared to NPC-43 and NPC-53 (Fig. 3 b). The induction of MHC-I potentially suggests improved tumour cell recognition that leads to antigen-specific cytolysis. Furthermore, CD8⁺ T cells derived from multiple donors consistently demonstrated antigen-specific killing, confirming the reproducibility of this response across individuals (sFig3). NLRC5 knockdown reduces MHC-I expression in NPC cells without impairing T cell-mediated cytotoxicity NLRC5 is an important regulator of MHC-I expression. To assess the effect of NLRC5 on MHC-I expression and T cell-mediated cytotoxic efficiency, we used siRNA to knockdown NLRC5 in NPC-43. NLRC5 knockdown in NPC-43 cells was validated at the gene level, showing a 76% reduction in NLRC5 mRNA expression, which remained low for up to 72 hours (Fig. 5 a). This knockdown was further confirmed at the protein level, which also resulted in a decrease in both NLRC5 and MHC-I expression (Fig. 5 b). Interestingly, while NLRC5 knockdown markedly reduced MHC-I expression, it did not affect the expression of other key MHC-I associated genes such as B2M and CD274 , but led to an increase in TAP1 expression (Fig. 5 c). Despite reduced MHC-I expression following NLRC5 knockdown, no significant difference in tumour cell killing between control and siNLRC5-treated cells was observed (Fig. 5 d). This is likely due to the restoration of NLRC5 and MHC-I expression upon co-culture with peptide-trained T cells (Fig. 5 E). These findings suggested that vaccine can upregulate both NLRC5 and MHC-I expressions thereby enhancing target cell susceptibility to T cell-mediated cytotoxicity. Vaccine-induced upregulation of NLRC5 enhances antigen presentation and suppresses tumour growth in vivo Due to the lack of syngeneic NPC mouse model, we employed the B16F10-A2 mouse melanoma characterized by poor immunogenicity and low MHC-I expression to evaluate the efficacy of the DNA vaccine in an antigen presentation-deficient setting ( 35 ). Although limited to a single model, our in vivo efficacy study demonstrated that a prime-boost regime of the DNA vaccine significantly suppressed tumour growth compared to the pLDOM control animals (Fig. 6 a). Vaccine-treated tumours also demonstrated a significant increase in CD8 + T cell infiltration as compared to control animals (Fig. 6 b), suggesting enhanced antitumor immunity in poorly immunogenic B16F10 tumours. To further investigate whether vaccine modulates antigen presentation, we performed RNA sequencing on the tumour samples. Our KEGG pathway enrichment analysis identified antigen processing and presentation as one of the most significantly upregulated pathways following DNA vaccination (Fig. 6 c). This was supported by the overall upregulation of genes of the APM signature. In particular, Nlrc5 , Tap1 and Rfx5 were the genes significantly increased in the DNA vaccine-treated group (Fig. 6 d). The in vivo findings align with our in vitro data, where co-culture of NPC cells with peptide-trained T cells upregulated NLRC5, TAP1, and B2M, restored MHC-I expression, and enhanced antigen-specific cytotoxicity. Together, these results demonstrate that the vaccine can upregulate NLRC5 and promote antigen presentation, thereby strengthening anti-tumour immune responses. Discussion Cancers can evade immune surveillance by downregulating APM, which in turn impairs antigen presentation and reduces immunogenicity. In about 30% of NPC cases with APM downregulation, somatic alterations such as mutations and structural variants affecting MHC class I expression were found in NLRC5, HLA-A, and HLA-B, occurring in 2.9 to 8.5 percent of cases ( 10 ). This observation suggested that only a small subset of APM-deficient tumours may represent hard lesions with irreversible genetic changes, while others may be soft lesions with potentially reversible suppression. In this study, we identified that APM downregulation in NPC may arise from the transcriptional downregulation of NLRC5 , a key transcriptional regulator of MHC-I expression ( 36 ). Indeed, we showed that NLRC5 expression is strongly correlated with MHC-I expression in NPC. NLRC5 is critical for mediating MHC-I-dependent CD8 + T cell antitumor immunity ( 37 ). Studies have shown that NPC patients with MHC-I downregulation exhibit reduced CD8 + T cell populations in both tumours and peripheral blood compared to healthy controls, indicating impaired CD8 + T cell-mediated antitumor immunity ( 11 ). Furthermore, low NLRC5 expression is associated with poor patient survival, underscoring its clinical relevance ( 37 , 38 ). MAGED4B and FJX1 are shared TAAs and were found to be upregulated in 91% of NPC cases, consistent with the 95% reported in head and neck cancer ( 25 ). These TAAs contribute to oncogenesis by promoting cancer cell proliferation and invasion ( 25 , 39 , 40 ). Importantly, MAGED4B and FJX1 are reported to be immunogenic, and their high expression in NPC highlights their potential as viable targets for vaccine-based therapies. However, 60% of NPC cases overexpressing these TAAs exhibit downregulation of MHC-I. This paradox underscores a critical therapeutic opportunity: although TAAs are abundantly expressed, their recognition by cytotoxic T cells is compromised. Therefore, restoring MHC-I-mediated antigen presentation is essential to enhance immune recognition. Here, we demonstrate that a cancer vaccine approach can effectively restore antigen presentation and enhance immunogenicity in APM-downregulated NPC. Our in vitro experiments demonstrated that co-culturing peptide-trained T cells with NPC cell lines induced the expression of NLRC5 and MHC-I across all three NPC cell lines, along with key MHC-I-associated genes, TAP 1 and B2M . While a potent induction of NLRC5 could be achieved through IFNγ stimulation, the clinical use of systemic IFNγ is limited due to its association with severe and potentially life-threatening toxicities ( 41 ). Alternatively, the FDA-approved HDAC inhibitor SAHA has been shown to enhance MHC-I expression by reversing epigenetic silencing in various cancer models ( 42 – 44 ). However, in our study, SAHA exhibited limited efficacy in the NPC cells. Only NPC-43 showed dose-dependent induction of MHC-I, while NPC-53 and NPC-268 displayed minimal response even at the highest concentration tested. Although HDAC status in NPC cell lines is not known, we postulated that HDAC-mediated epigenetic suppression may not be the underlying mechanism of MHC-I downregulation in these NPC cell lines tested. This upregulation of NLRC5 and MHC-I protein expression by peptide-trained T cells enhanced tumour immunogenicity and promoted antigen-specific cytotoxicity against NPC cell lines. To further investigate the role of NLRC5, we performed knockdown experiments in NPC cells. Although NLRC5 deficiency led to reduced MHC-I expression, it was reversed upon co-culture with peptide-trained T cells, resulting in T cell-mediated cytotoxicity. Consistently, our in vivo studies showed that antigen-specific DNA vaccination enhances antitumor immunity in a MHC-I downregulated antigen-expressing B16F10-A2 melanoma syngeneic mouse model. Notably, tumour suppression was accompanied by increased CD8 + T cell infiltration following vaccination. Consistent with our in vitro study, the APM pathway was among the most significantly upregulated pathways in DNA vaccine-treated tumours. Among the 17-gene APM signature, Nlrc5 was found to be significantly upregulated in mice responding to DNA vaccination. The induction and stable expression of Nlrc5 could be crucial for MHC-I upregulation, which further restores immunogenicity in B16F10 melanoma cells ( 33 , 36 , 45 , 46 ). Hence, we postulated that peptide-trained T cells naturally secrete inflammatory cytokines such as IFNγ and TNFα, which activate JAK-STAT signalling to induce NLRC5 transcription ( 47 ). NLRC5, in turn, binds to promoter regions to enhance the transcription of MHC-I, B2M, and TAP1, thereby promoting antigen presentation ( 48 , 49 ). Additionally, others have shown that NLRC5 can also regulate IFNγ response, providing a feedback loop between NLRC5 and IFNγ that sustains MHC-I upregulation ( 33 ). Strategies aimed at inducing IFNγ could potentially enhance APM expression. However, it is important to consider that while IFNγ is crucial for initiating immune responses, excessive IFNγ stimulation may induce CD274 in the absence of specific antigen recognition and lead to T cell dysfunction ( 50 ). Therefore, approaches that rely on antigen-specific activation, such as cancer vaccines, may offer a more targeted and clinically feasible strategy for restoring antigen presentation in NPC. Moreover, it is also worth noting that MHC-I expression can be regulated through alternative pathways, such as NF-κB and cGAS-STING signaling ( 33 ), which were not examined in this study. Nevertheless, our findings highlight the potential of vaccination strategies to counteract transcriptional suppression of APM genes and overcome immune evasion mechanisms in NPC. NPC is characterized by abundant immune cell infiltration and high CD274, yet its response rate to ICIs remains limited compared to other "hot" tumours. One contributing factor is the downregulation of APM, including MHC-I ( 6 , 51 ). Restoring MHC-I expression has been shown to improve tumour sensitivity to ICI treatment ( 50 , 52 ). In our study, peptide-trained T cells robustly induced MHC-I-associated components in NPC cells, and only minimally upregulated CD274. This is notable because PD-L1 expression is often co-regulated with MHC-I in response to IFNγ. The modest induction of CD274 alongside strong MHC-I upregulation suggests that vaccination may selectively enhance tumour immunogenicity without triggering excessive immune checkpoint expression. Our study has several limitations that require further investigation. This study suggests that cancer vaccines may enhance immune recognition in tumours with transcriptionally suppressed MHC-I. However, MHC-I downregulation may also result from enhanced protein degradation and disruptions in endocytic trafficking, which are not explored in the current study and whose responsiveness to cancer vaccination remains unknown. Due to the lack of a syngeneic NPC mouse model, in vivo experiments were performed using the B16F10 melanoma model. Although this model may not fully recapitulate the NPC tumour microenvironment, it served as a useful platform to confirm APM modulation following vaccination. These limitations underscore the need for employing more representative NPC models and comprehensive analyses of MHC-I regulatory pathways to better understand immune evasion and optimize immunotherapeutic strategies in NPC. Conclusions Our study demonstrates that peptide-trained T cells can upregulate MHC-I expression in NPC cells via NLRC5 transcriptional regulation, enhancing immune recognition and cytotoxic killing of tumour cells both in vitro and in vivo . These findings highlight the potential of cancer vaccines in APM-downregulated tumours, presenting a potential strategy to enhance immunotherapy efficacy, particularly for patients with MHC-I downregulation and resistance to current treatments. Abbreviations NPC Nasopharyngeal carcinoma ICI Immune checkpoint inhibitor NLRC5 NOD-like receptor family CARD domain containing 5 APM Antigen presentation machinery MHC-I Major histocompatibility complex class I MAGED4B MAGE family member 4B FJX1 Four-jointed box kinase 1 TAA Tumour-associated antigen IFNγ Interferon gamma SAHA Suberoylanilide hydroxamic acid CIITA Class II major histocompatibility complex transactivator TAP1 Transporter associated with antigen processing 1 B2M Beta-2-microglobulin Declarations Acknowledgements We would like to thank Touchlight Genetics Ltd for generously providing the doggybone DNA vaccine and for their valuable input and discussions throughout the course of this project. This study also utilised NPC cell lines generously provided by Translational Cancer Biology Research Unit, Cancer Research Malaysia. Funding This work was supported by the Technology Development Fund 2, TeD2 (TEF 04221148) from the Ministry of Science Technology and Innovation (MOSTI), Malaysia, Touchlight Genetic Ltd, United Kingdom and internal funding from Cancer Research Malaysia. Authors information Chai Phei Gan and Sau Yee Kok contributed equally to this work. Authors and Affiliations Cancer Immunology & Immunotherapy Research Unit, Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia. Chai Phei Gan & Sau Yee Kok & Natasha Zulaziz & Sok Ching Cheong & Kue Peng Lim Department of Mathematical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Bernard Kok Bang Lee Molecular & Clinical Cancer Medicine, University of Liverpool, UK Natalia Savelveya Contributions Conception and design, CPG, SYK SCC, and KPL; Experiment performance; CPG, SYK, NZ, and KPL; Data analysis and interpretation, CPG, SYK, BKBL, and LKP; Manuscript preparation, CPG, SYK, BKBL, and KPL. Manuscript revision, CPG, SYK, BKBL, NZ, SCC, NS and KPL. All authors contributed, studied, and approved the final manuscript. Corresponding author Kue Peng Lim Ethics declarations Ethics approval and consent to participate This work was approved by the Medical Ethics Committee of Penang General Hospital (NMRR-09-944-4848) and Faculty of Dentistry, University of Malaya [DF OS 2101/0002/2179(L). Informed consent was obtained from all donors. The animal study was approved by Experimental Animal Ethics Committee, National University Malaysia (CRM/2020/KUE PENG/25-MAR./1093-APR.-2020-APR.-2023). Consent for publication All authors approved the contents of the manuscript and agreed with the publication. Competing interests KPL and NS received funding from Touchlight Genetics Ltd for works in the development of cancer vaccine. The other authors declare no competing interests. Data availability Publicly available data used in this study were obtained from the NCBI Gene Expression Omnibus (GEO) under accession number GSE68799. The RNA-seq data generated in this study are not publicly available due to ongoing related studies but are available from the corresponding author upon reasonable request. References Cantù G. Nasopharyngeal carcinoma. A different head and neck tumour. Part A: from histology to staging. Acta Otorhinolaryngol Ital. 2023;43:85–98. Lee AWM, Ng WT, Chan LLK, Hung WM, Chan CCC, Sze HCK, et al. 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Supplementary Files SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplementaryTable3.xlsx sFig1202507.pdf sFig2202507.pdf sFig3202507.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 05 Jul, 2025 First submitted to journal 03 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7037136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485290332,"identity":"5f940167-9768-41bb-a58d-b4e71db20b6c","order_by":0,"name":"Chai Phei Gan","email":"","orcid":"","institution":"CRM: Cancer Research Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Chai","middleName":"Phei","lastName":"Gan","suffix":""},{"id":485290333,"identity":"5dd9337d-2e82-433d-a0dd-5ea112312832","order_by":1,"name":"Sau Yee Kok","email":"","orcid":"","institution":"CRM: Cancer Research Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Sau","middleName":"Yee","lastName":"Kok","suffix":""},{"id":485290334,"identity":"5c5b2981-63b5-4d01-8486-4f0300f286b7","order_by":2,"name":"Bernard Kok Bang Lee","email":"","orcid":"","institution":"Universiti Kebangsaan Malaysia Fakulti Sains dan Teknologi","correspondingAuthor":false,"prefix":"","firstName":"Bernard","middleName":"Kok Bang","lastName":"Lee","suffix":""},{"id":485290335,"identity":"3f98c9b2-0ff8-4b4a-a624-f4333fd56c7e","order_by":3,"name":"Natasha Zulaziz","email":"","orcid":"","institution":"CRM: Cancer Research Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Natasha","middleName":"","lastName":"Zulaziz","suffix":""},{"id":485290336,"identity":"9a4544d3-5e3c-4ff6-938e-7579f2dbd3bf","order_by":4,"name":"Sok Ching Cheong","email":"","orcid":"","institution":"CRM: Cancer Research Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Sok","middleName":"Ching","lastName":"Cheong","suffix":""},{"id":485290337,"identity":"e006676c-d4a8-48be-ab5d-3eaab1f26901","order_by":5,"name":"Natalia Savelveya","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Savelveya","suffix":""},{"id":485290338,"identity":"ff18d5ba-8d3d-46af-937a-bc3ee0a75a34","order_by":6,"name":"Kue Peng Lim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABJUlEQVRIiWNgGAWjYBACCRiDj5mB8QEOORzCbMwMzAYkagEiCRxyqEByRvKxBx931DKwsXOnVfyouSPPL3324cMfFXb5/LMbWDfzYGqRlkhLN5x55jjQYbzbbvYce2Y4sy/d2JjnTLLljDsH2G5j0SInkWMmzdt2DKzlNgPb4QSDM2xs0oxtzAYGEgk4tOR/k/4L1VLM8O9wgv0ZNvafP//V49QiLZEDMrMGrIWZsQ1oCw8bGwNvw2GcWiR7npkb9rYd4AFq2SzZ23fYcMYZNmZpnmPHDSRuJLbdnIOpReJ48rMHP9vq5Pj5z2788OPbYXn+HjbGjz9qqg34gYF54w3WcAbGCMNhLA5gYGBsYMIqDtZSh1UGqOkHDolRMApGwSgYSQAAhpxeuFCrvkAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5670-8070","institution":"Cancer Research Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Kue","middleName":"Peng","lastName":"Lim","suffix":""}],"badges":[],"createdAt":"2025-07-03 10:41:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7037136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7037136/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-07418-x","type":"published","date":"2025-12-23T15:58:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87267482,"identity":"71616813-36e2-4bdd-bc68-e56bbb1ff4a4","added_by":"auto","created_at":"2025-07-22 08:06:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323399,"visible":true,"origin":"","legend":"\u003cp\u003eAntigen presentation machinery (APM) is downregulated in NPC. \u003cstrong\u003eA\u003c/strong\u003e Heatmap of APM-related genes from GSE68799, showing z-score normalised expression in NPC tumours compared to non-NPC tissues. The two columns on the right represent the average z-scores across all non-NPC and tumour samples. The analysis highlights widespread downregulation of APM-related genes in tumours. Asterisks denote statistically significant differential expression (*p \u0026lt; 0.05, **p \u0026lt; 0.01). \u003cstrong\u003eB\u003c/strong\u003e Correlation analysis reveals that \u003cem\u003eNLRC5\u003c/em\u003e downregulation is strongly and positively associated with reduced expression of MHC-I genes (\u003cem\u003eHLA-A\u003c/em\u003e, \u003cem\u003eHLA-B\u003c/em\u003e, and \u003cem\u003eHLA-C\u003c/em\u003e) in NPC.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/976ba61549bbc7ec4bbe4680.jpg"},{"id":87269385,"identity":"aed3fd2b-8f85-4617-944c-c24db80a6574","added_by":"auto","created_at":"2025-07-22 08:14:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":623485,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of MHC-I downregulation in NPC by IHC staining. \u003cstrong\u003eA\u003c/strong\u003e Representative micrograph of MHC-I and MHC-II staining in NPC and non-NPC tissues. Insets show 20x magnification of the selected regions. \u003cstrong\u003eB\u003c/strong\u003e Proportion of samples with low MHC expression. In comparison to the control, 63% of NPC exhibited low MHC-I, while 29% exhibited low MHC-II\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/628f607c007aa6785c379820.jpg"},{"id":87269382,"identity":"046e11b1-07aa-4c7f-aa1b-ddf0e8c40d60","added_by":"auto","created_at":"2025-07-22 08:14:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":462502,"visible":true,"origin":"","legend":"\u003cp\u003eModulation of MHC-I expression in NPC cells by IFNγ, peptide-trained T cells, and HDAC inhibitor. \u003cstrong\u003eA\u003c/strong\u003e Surface expression of HLA-ABC on NPC cell lines measured by median fluorescent intensity (MFI) of PE-tagged HLA-ABC at baseline (left), and the induction of HLA-ABC by IFNγ treatment detected by Western Blotting (right). \u003cstrong\u003eB\u003c/strong\u003e Enhancement of HLA-ABC expression by peptide-trained T cells against MAGED4B (M-T) and FJX1 (F-T) after co-culture (left); bar chart shows relative changes of HLA-ABC compared to control cells grown in culture media (CM; right). \u003cstrong\u003eC\u003c/strong\u003e Western blot analysis of total HLA-ABC expression following SAHA treatment. \u003cstrong\u003eD\u003c/strong\u003e Fold change in surface HLA-ABC expression relative to the control cells grown in CM.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/afeae2a1739dafe8c422dc2b.jpg"},{"id":87269381,"identity":"acbaba35-ceef-4299-a204-7d28fdf6597e","added_by":"auto","created_at":"2025-07-22 08:14:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":432082,"visible":true,"origin":"","legend":"\u003cp\u003ePeptide-trained T cells enhance MHC-I-associated gene expression and promote antigen-specific cytotoxicity in NPC cells. \u003cstrong\u003eA\u003c/strong\u003e Peptide-trained T cells significantly upregulated \u003cem\u003eNLRC5\u003c/em\u003e, \u003cem\u003eTAP1\u003c/em\u003e and \u003cem\u003eB2M\u003c/em\u003e, with minimal induction of \u003cem\u003eCD274\u003c/em\u003e (PD-L1) compared to IFNγ treatment. \u003cstrong\u003eB\u003c/strong\u003e Co-culture of peptide-trained T cells (effector) with NPC cells (target) at a 10:1 effector-to-target ratio induced antigen-specific killing. ORL-195/MAGED4B which overexpresses MAGED4B served as positive control, while ORL-195LacZ, lacking MAGED4B expression served as a negative control. Cytolysis below 20% was considered background noise, indicating no antigen-specific killing.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/8b10661eda88642635728138.jpg"},{"id":87269384,"identity":"6dd53727-b3c1-4ba6-a1c5-1c8ede50cacb","added_by":"auto","created_at":"2025-07-22 08:14:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":351331,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of NLRC5 knockdown on MHC-I expression and antigen-specific killing of NPC-43.\u003cstrong\u003e A\u003c/strong\u003e \u003cem\u003eNLRC5\u003c/em\u003e gene expression after siRNA treatment. \u003cstrong\u003eB\u003c/strong\u003e Western blot showing reduced MHC-I protein levels after NLRC5 knockdown. \u003cstrong\u003eC\u003c/strong\u003e Relative expression of MHC-I-associated genes in NLRC5 knockdown cells compared to control cells. Dotted lines indicate baseline levels in control cells. \u003cstrong\u003eD\u003c/strong\u003e No reduction in cytolysis after NLRC5 knockdown compared to control cells, which endogenously express NLRC5. \u003cstrong\u003eE \u003c/strong\u003ePeptide-trained T cells restore both NLRC5 and MHC-I expressions in NLRC5 knockdown cells.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/153d06a1bad1e4b0977c71d9.jpg"},{"id":87267484,"identity":"24e6f3d3-4e1b-46bd-b8f3-8b5b0f832013","added_by":"auto","created_at":"2025-07-22 08:06:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":408928,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of tumour growth by DNA vaccine targeting MAGED4B and FJX1 via upregulation of APM genes in a syngeneic mouse model.\u003cstrong\u003e A\u003c/strong\u003eThe subcutaneous tumour volume was significantly reduced in mice treated with the DNA vaccine compared to the control group. \u003cstrong\u003eB\u003c/strong\u003e Immune cell infiltration into the tumour increased after DNA vaccination. Insets show 20x magnification of the selected regions. \u003cstrong\u003eC\u003c/strong\u003e KEGG pathway analysis revealed that APM pathway is upregulated in DNA vaccine-treated group. \u003cstrong\u003eD\u003c/strong\u003e Heatmap showing gene expression in the 17-gene APM signature. The two columns on the right represent the average control and DNA vaccine samples, respectively. Asterisks denote statistically significant differential expressions (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/0cf673b0dcfba42daebe13e7.jpg"},{"id":99172408,"identity":"5437d5a8-fa6e-445b-a639-419443b84075","added_by":"auto","created_at":"2025-12-29 16:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3800374,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/8fababfe-8440-416f-9310-a92c689402f4.pdf"},{"id":87269380,"identity":"44a855ad-2eef-4ee7-abd6-9365cb7f58a5","added_by":"auto","created_at":"2025-07-22 08:14:52","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":10973,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/c623d79d7f6a1d52f5ef1df1.xlsx"},{"id":87267487,"identity":"82cc9c4f-31c1-4587-9694-30f88b96aca3","added_by":"auto","created_at":"2025-07-22 08:06:52","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":11008,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/31896ba61aba5916b9742898.xlsx"},{"id":87267492,"identity":"7f8f34c1-93fa-4c39-ac5e-ca115db9397f","added_by":"auto","created_at":"2025-07-22 08:06:52","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":9343,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/cff8ab669fa22a0a4224511d.xlsx"},{"id":87270751,"identity":"da41ecb7-f6b8-4f5f-ba28-e48ec1bf77ef","added_by":"auto","created_at":"2025-07-22 08:22:52","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":159352,"visible":true,"origin":"","legend":"","description":"","filename":"sFig1202507.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/45669029df3693280053fbd8.pdf"},{"id":87267503,"identity":"447a07df-5808-4e4e-a31b-6126edec82a6","added_by":"auto","created_at":"2025-07-22 08:06:53","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":116288,"visible":true,"origin":"","legend":"","description":"","filename":"sFig2202507.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/69024a3bb37fdd05a5f03b35.pdf"},{"id":87269386,"identity":"385fcc80-9b32-4f04-bf2a-7c371d553be1","added_by":"auto","created_at":"2025-07-22 08:14:52","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":137122,"visible":true,"origin":"","legend":"","description":"","filename":"sFig3202507.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7037136/v1/c0da8688ca9e927fac96a62c.pdf"}],"financialInterests":"","formattedTitle":"Cancer vaccine overcomes immune evasion of nasopharyngeal carcinoma by restoring MHC-I through transcriptional regulation of NLRC5","fulltext":[{"header":"Background","content":"\u003cp\u003eNasopharyngeal carcinoma (NPC) is a subset of head and neck cancer, and exhibits a distinct population-specific prevalence, occurring nearly 40 times more frequently in Asians than in Caucasians (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Due to its non-specific early symptoms, over 80% of patients are diagnosed at a locally advanced stage or with distant metastasis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). While early and locally advanced NPC respond well to radiotherapy, recurrent or metastatic NPC carries a poor prognosis, with a median overall survival of 20 months (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Immune checkpoint inhibitors (ICIs) are increasingly used to treat patients with recurrent or metastatic disease, considering that NPC is an immune hot tumour characterized by its association with Epstein-Barr virus (EBV) infection, dense lymphocytic infiltration and elevated PD-L1 expression (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, overall response rates to anti\u0026ndash;PD-1 monotherapy remain modest at 10\u0026ndash;43% and fail to produce a durable clinical benefit (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). One proposed reason for this limited efficacy is the deficiency in antigen processing and presentation machinery (APM), which is essential for effective T cell-mediated anti-tumour immunity (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDownregulation of the APM pathway has been reported in 30\u0026ndash;80% of NPC patients (\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Specifically, the expression of major histocompatibility complex (MHC-I) molecules, including the HLA-A and HLA-B, as well as their associated components such as TAP1 and TAP2, is significantly reduced in NPC compared to normal tissue. APM downregulation can result from \u0026ldquo;hard/irreversible\u0026rdquo; genomic alterations, such as chromosomal structural variants, single-nucleotide variants and mutations, which lead to inactivation of HLA-A and HLA-B in 13% and 9% of NPC, respectively (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). \u0026ldquo;Soft/reversible\u0026rdquo; alterations driven by epigenetic, transcriptional and posttranscriptional mechanisms may also contribute to APM downregulation, although their prevalence in NPC remains unclear (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Additionally, EBV-encoded proteins such as BNLF2a and BILF1 can also cause APM downregulation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). MHC-I downregulation is associated with poorer survival outcome, likely due to compromised antigen presentation, enabled cancer cells to evade immune detection (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent research has demonstrated the potential of targeting APM to overcome immune escape in cancer by promoting tumour antigenicity, enhancing immunogenicity, and stimulating T cell-mediated tumour killing. For example, platinum-based chemotherapies have been shown to upregulate MHC-I on both tumours and dendritic cells in preclinical studies, thereby improving the efficacy of anti-PD1 thereapy (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In NPC, combining anti-PD1 therapy with gemcitabine and cisplatin has led to improved response rates of 70\u0026ndash;91% (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), likely due to chemotherapy-induced immune modulation. This effect may be mediated by increased MHC-I antigen presentation, potentially through activation of pathways such as IFNβ and NF-κB (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, despite these immunostimulatory effects, the combination of chemotherapy and anti-PD1 is associated with substantial toxicity, with grade\u0026thinsp;\u0026ge;\u0026thinsp;3 adverse events reported in 57\u0026ndash;94% of NPC patients. These limitations underscore the need for alternative strategies that can enhance antigen presentation with fewer side effects. Cancer vaccines targeting tumour-associated antigens (TAAs) offer a promising approach to provoke an immune response by priming tumour-reactive T cells against existing tumours (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Notably, MAGED4B and FJX1 are highly immunogenic TAAs that are overexpressed in NPC (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Vaccines targeting these TAAs significantly inhibited tumour growth when combined with anti-PD1 therapy (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In this study, we investigated the potential of cancer vaccines targeting MAGED4B and FJX1 in modulating APM to restore anti-tumour immunity in NPC. Specifically, we demonstrated that co-culturing peptide-trained T cells and NPC cells endogenously expressing these TAAs enhances antigen presentation through the upregulation of NOD-like receptor family CARD domain containing 5 (NLRC5) and MHC-I in the APM pathway to induce T cell-mediated cytotoxicity.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eBioinformatics analyses of APM gene expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe RNA sequencing dataset GSE68799, comprising 41 NPC and 4 non-NPC tissues, was obtained from the Gene Expression Omnibus database. This dataset was selected for its large number of tumour samples and its accessibility through a public repository. A curated APM signature consisting of 17 genes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) was used to identify differentially expressed genes (DEGs) between NPC and non-NPC tissues using DESeq2, with thresholds set at |log2 fold change| \u0026gt;1 and \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). A heatmap illustrating gene expression patterns was generated using the Morpheus web tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.broadinstitute.org/morpheus\u003c/span\u003e\u003cspan address=\"https://software.broadinstitute.org/morpheus\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). APM genes that were significantly downregulated in NPC tissues were further analysed using Pearson correlation to assess their association with MHC-I genes (HLA-A, HLA-B, and HLA-C). Additionally, the KEGG antigen processing and presentation pathway was analysed in the GSE68799 dataset to assess the prevalence of HLA gene downregulation in NPC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFormalin-fixed, paraffin-embedded (FFPE) tissues were used to assess target protein expression via immunohistochemistry (IHC) staining with the EnVision\u0026thinsp;+\u0026thinsp;system (Dako, Germany). These included whole tissue sections from tumours (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14) and a tissue microarray (NH1001a; TissueArrays.com, USA) comprising tumour samples (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21) and non-NPC tissues (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5). The whole tissue sections were obtained from Penang General Hospital upon medical ethics approval (NMRR-09-944-4848). FFPE tissue sections were deparaffinized and dehydrated in decreasing grades of ethanol. Antigen retrieval was performed by boiling the tissues in the respective antigen retrieval buffer in the microwave. The Tris/EDTA pH 9.0 buffer was used for antigen retrieval of FJX1, while sodium citrate pH 6.0 buffer was used for antigen retrieval of MHC-I, MHC-II and MAGED4B. Tissues were incubated with 1% bovine serum albumin in Tris-buffered saline to prevent unspecific binding. The primary antibodies were anti-HLA Class-I monoclonal antibody, clone EMR8-5 (1:100; Abcam, USA), anti-HLA Class-II monoclonal antibody clone 6C6 (1:100; Abcam, USA), anti-MAGED4B (1:100; Sigma-Aldrich, USA) and anti-FJX1 (1: 200; Sigma-Aldrich, USA). After peroxidase blocking, antigen expressions were visualized using the Envision\u0026thinsp;+\u0026thinsp;Dual Link System HRP (DAB+) kit (Dako, Germany).\u003c/p\u003e\u003cp\u003eThe immunoreactivity of MHC-I was evaluated based on the criterion established by the Human Leukocyte Antigen and Cancer component of the 12th International Histocompatibility Workshop (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The staining intensity of MHC-I in the lymphocytes and vessel endothelium is regarded as internal positive controls. Tumour cells stained as similar or greater intensity than the internal positive control was graded, and area positivity score was given as 0 (no expression): less than 25%; 1 (low expression): 25 to 75% and 2 (high expression): greater than 75%. On the other hand, the immunoreactivity of MAGED4B and FJX1 was scored based on a 4-point intensity scoring system: 0\u0026thinsp;=\u0026thinsp;negative expression; 1\u0026thinsp;=\u0026thinsp;weak positive; 2\u0026thinsp;=\u0026thinsp;moderate positive; 3\u0026thinsp;=\u0026thinsp;strong positive. Score 0 and 1 were grouped as low expression, 2 and 3 were grouped as high expression, as reported previously (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture and cell transfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNPC-43 and NPC-53 cells, derived from recurrent tumours (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and NPC-268 cell derived from a primary tumour (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) were cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), 100 IU Penicillin/Streptomycin (Gibco, USA) and 4 \u0026micro;M Rock inhibitor (Y-27632; Enzo Life Sciences, USA). Cells were maintained at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. NPC cell lines used in this study were authenticated by short tandem repeat DNA profiling to confirm their identity and rule out cross-contamination. Additionally, they were tested and confirmed to be mycoplasma-free.\u003c/p\u003e\u003cp\u003eTo knockdown NLRC5 expression, NLRC5 siRNA (5\u0026rsquo; GGA CAC CUG GCA GUC UUU CAU UCA U3\u0026rsquo;; Invitrogen, USA) was transfected into NPC cells using Lipofectamine\u0026trade; RNAiMAX (Invitrogen, USA) according to the manufacturer\u0026rsquo;s protocol. For control, cells were transfected with non-targeting (NT) siRNA (Invitrogen, USA). To evaluate the transfection efficiency of NLRC5 knockdown, cells were harvested at the indicated time points for real-time PCR and western blot assays.\u003c/p\u003e\u003cp\u003eThe mouse melanoma cell line B16F10 expressing the human-HLA-A2 gene (B16F10-A2) was cultured in RPMI-1640 medium (Gibco, USA) containing 10% FBS and 100 IU Penicillin/Streptomycin (Gibco, USA), at 37\u0026deg;C in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. To express MAGED4B and FJX1 in B16F10-A2 cells, the pLENTI-6.3 plasmids carrying MAGED4B and FJX1 were simultaneously transfected into the cells using Lipofectamine according to manufacturer\u0026rsquo;s instruction. Cells were further selected using antibiotics (G418 and blasticidin) for one month. Western blotting was performed to confirm the expression of MAGED4B and FJX1 in B16F10-A2 prior to their use in the mouse experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneration of MAGED4B- and FJX1-specific T cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were harvested from blood samples collected into cellular preparation tubes (Becton Dickinson, USA) from three healthy donors (HD30, HD31 and HD32). Red blood cells were removed using Ammonium Chloride Potassium (Gibco, USA) lysing buffer. The use of peripheral blood samples in this study was approved by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [DF OS 2101/0002/2179(L).\u003c/p\u003e\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells were isolated through negative selection using the human CD8\u003csup\u003e+\u003c/sup\u003e T cell isolation kit (Miltenyi Biotec, Germany). Both CD8\u003csup\u003e+\u003c/sup\u003e and non-CD8\u003csup\u003e+\u003c/sup\u003e cell fractions were collected, with the non-CD8\u003csup\u003e+\u003c/sup\u003e fraction serving as autologous antigen-presenting cells (APC). The CD8\u003csup\u003e+\u003c/sup\u003e T cells were cultured in RPMI medium supplemented with 10% FBS and 30ng/ml anti-CD3 (OKT3 clone, Miltenyi Biotec, Germany). \u003cem\u003eA\u003c/em\u003entigen-specific T cells were generated using a modified protocol adapted from Erskine \u003cem\u003eet al.\u003c/em\u003e 2012 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). CD8\u003csup\u003e+\u003c/sup\u003e T cells were stimulated with 10\u0026micro;g/mL of overlapping peptide pools derived from MAGED4B (M-OPP) and FJX1 (F-OPP; JPT Peptide Technologies, Germany), respectively. CD8\u003csup\u003e+\u003c/sup\u003e T cells were supplemented with 50 IU/mL of recombinant human interleukin-2 (IL-2; R\u0026amp;D Systems, USA) from day 3 onwards. Restimulation with autologous APC cells was performed at day 7 and 14. To prepare APCs, the non-CD8\u003csup\u003e+\u003c/sup\u003e fraction was pulsed with 10 \u0026micro;g/mL of M-OPP or F-OPP, followed by mitomycin C treatment to inhibit cell proliferation. CD8⁺ T cells that completed 3 rounds of stimulation were hereafter referred to as peptide-trained T cells and were used in subsequent functional assays.\u003c/p\u003e\u003cp\u003e\u003cb\u003eModulation of APM gene expression by peptide-trained T cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression of APM genes in NPC cell lines following exposure to peptide-trained T cells was assessed \u003cem\u003ein vitro\u003c/em\u003e. NPC cells (target) were seeded overnight at 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e in a 6-well plate, then co-cultured with peptide-trained T cells (effector) at an effector-to-target ratio of 1: 10 for 24 hours. NPC cells stimulated with 100 ng/mL IFNγ (R\u0026amp;D Systems, USA) served as a positive control.\u003c/p\u003e\u003cp\u003eFollowing incubation, total RNA was extracted from NPC cell lines using RNeasy Micro kit (Qiagen, Germany), and cDNA was synthesized from 1\u0026micro;g of total RNA using BIOLINE SensiFAST reagent kit (Bioline Reagent Ltd, UK). Gene expression analysis was performed on the 7500 Real-Time PCR System (Applied Biosystems, USA) using specific PCR primers (Supplementary Table\u0026nbsp;1) and Power Sybr Green PCR Master Mix (Applied Biosystems, USA). Relative mRNA levels of \u003cem\u003eNLRC5, CD274, TAP1\u003c/em\u003e, and \u003cem\u003eB2M\u003c/em\u003e were obtained after normalisation to \u003cem\u003eGAPDH\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression of human leukocyte antigen ABC (HLA-ABC), corresponding to MHC-I was analysed by flow cytometry. Firstly, HLA-A typing was performed on PBMCs and NPC cell lines using specific antibodies and isotype controls. Briefly, cells were stained with FVS780 viability dye (BD Biosciences, USA) for 15 minutes followed by incubation with the respective primary antibodies: PE-conjugated mouse anti-human HLA-A2 (Clone BB7.2, BD Biosciences, USA), unconjugated HLA-A11 +/-A3 IgG (One Lambda, USA), unconjugated HLA-A2 and A24 IgM (One Lambda, USA), and unconjugated HLA-A24, A11, A2403\u0026thinsp;+\u0026thinsp;IgM (One Lambda, USA). For isotype controls, cells were stained with mouse IgG2b-UNLB (SouthernBiotech, USA) and mouse IgM-UNLB (SouthernBiotech, USA). Cells stained with PE-conjugated mouse anti-human HLA-ABC (Clone W6/32, BD Biosciences, USA, 567582) served as the positive control. Unconjugated primary antibodies and isotype controls were further incubated with the corresponding secondary antibodies: Goat F(ab')₂ anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), human ads-PE (SouthernBiotech, USA), and Goat F(ab')₂ anti-mouse IgM, human ads-PE (SouthernBiotech, USA). After staining, a minimum of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were analysed using the LSRFortessa\u0026trade; X-20 Cell Analyzer (BD Biosciences, USA), with gating against unstained and isotype controls.\u003c/p\u003e\u003cp\u003eTo assess total MHC-I expression on NPC cells, cells were stained with FVS780, followed by HLA-ABC staining, and analysed as described above. The median fluorescence intensity (MFI) of HLA-ABC was quantified using FlowJo software (BD Biosciences, USA) to determine the relative abundance of HLA-ABC among the NPC cell lines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNPC cell lines were lysed in RIPA buffer (Thermo Scientific, USA) supplemented with 1x Halt protease and phosphatase inhibitors cocktail (Thermo Scientific, USA). Protein concentrations were measured using Pierce BCA protein assay kit (Thermo Scientific, USA). Protein lysates were denatured by heating at 98\u0026deg;C for 10 minutes. Equal amounts of protein were separated on a 10\u0026ndash;12% SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk and probed with antibodies against MAGED4B (1:1000, HPA003554, Sigma-Aldrich, USA), FJX1 (1:1000, HPA059220, Sigma-Aldrich, USA), HLA Class I A/B/C (1:1000, clone EMR8-5, Ab70328, Abcam, USA), and NLRC5 (1:500, clone 3H8, MABF260, Millipore, USA). Anti-GAPDH antibody (1:1000, Proteintech, USA) and anti-tubulin (1:1000, T5168, Sigma-Aldrich, USA) were used as a loading control. An Immobilon Forte Western HRP substrate (Milipore, USA) was used for chemiluminescence detection and images taken using Western blot imager (Azure Biosystem, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSuberoylanilide hydroxamic acid (SAHA) treatment assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNPC cells were seeded at 3x10\u003csup\u003e5\u003c/sup\u003e cells per well in a 6-well plate overnight and serum-starved for 24 hours. Cells were treated with SAHA (Cayman Chemical, USA) at a final concentration of 1 \u0026micro;M, 5 \u0026micro;M and 10 \u0026micro;M for 24 hours in the CO\u003csub\u003e2\u003c/sub\u003e incubator. Subsequently, cells were harvested to assess MHC-I protein expression using HLA-ABC MFI quantification by flow cytometry and Western blotting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eT cell-mediated cytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cytotoxic activity of peptide-trained T cells against NPC cell lines was assessed using the xCELLigence Real-Time Cell Analyzer (RTCA; Agilent, USA). Briefly, background impedance values of 96 well E-Plate (Agilent, USA) filled with medium were obtained. NPC target cells were seeded into the E-plate (4x10\u003csup\u003e4\u003c/sup\u003e cells/well for NPC43 and NPC53 or 2x10\u003csup\u003e4\u003c/sup\u003e cells/well for NPC268), and allowed to adhere for 24 hours in the RTCA. peptide-trained T cells were added to target cells at a 10:1 effector-to-target ratio. Impedance values were recorded hourly for 24 hours. Cell index was normalized to the time of effector T cells addition, and cytolysis was calculated in relative to control cells without effector T cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAAD mice (transgenic for HLA-A2.1/H2-Dd allele) were bred and housed in the Animal Facility Centre of National University Malaysia. AAD mice (6\u0026ndash;10 weeks) were inoculated with 1 x10\u003csup\u003e5\u003c/sup\u003e B16F10-A2 cell line expressing MAGED4B/FJX1 and were randomised into 2 groups to receive (i) 40\u0026micro;g plasmid DOM (pLDOM, control); (ii) 40 \u0026micro;g each of doggybone DNA vaccine encoding MAGED4B/FJX1 (DNA vaccine); intramuscular aided by electroporation administration of DNA vaccine was given on day 5 and day 21 post cell inoculation; Tumour size was monitored 2 times/week until day 40. Tumour volumes were evaluated using the formula: volume = \u0026frac12; (length X width\u003csup\u003e2\u003c/sup\u003e). Animal study was approved by Experimental Animal Ethics Committee, National University Malaysia (CRM/2020/KUE PENG/25-MAR./1093-APR.-2020-APR.-2023).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA sequencing and bioinformatic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from mouse tumour tissue using the Qiagen RNeasy mini kit (Qiagen, Germany), with purity and integrity assessed via Nanodrop (Thermo Scientific, USA) and the RNA Nano 6000 Assay Kit run on the Bioanalyzer 2100 system (Agilent, USA). RNA library was prepared from 1 \u0026micro;g of total RNA using the NEBNext\u0026reg; UltraTM RNA Library Prep Kit (NEB, USA) and sequenced on Illumina NovaSeq (Illumina, USA) to generate paired-end 150bp reads. Raw reads (FASTQ format) were processed with fastp to remove adapter sequences, poly-N regions, and low-quality reads. Clean reads were mapped to the reference genome using HISAT2. DEGs between the DNA vaccine and control groups were identified using a joint filtering method, with thresholds for up-regulated and down-regulated genes set at |log\u003csub\u003e2\u003c/sub\u003e fold change| \u0026gt;1 and \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). To further analyse the functional significance of these DEGs, the KEGG pathway enrichment analysis was performed using Metascape (Broad Institute, USA) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). A heatmap of genes in the 17-gene APM signature was generated to examine the changes in gene expression between the DNA vaccine and control group.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses for bioinformatics studies are described in the respective sections. Experimental data were analysed using GraphPad Prism 10 (GraphPad Software, USA). IHC scoring of MHC-I expression in NPC tissues was evaluated using Fisher\u0026rsquo;s exact test to determine associations with clinical parameters. The expression of APM genes in NPC cells stimulated by peptide-trained T cells or IFNγ was compared to control cells using one-way ANOVA followed by Bonferroni\u0026rsquo;s post-hoc test for multiple comparisons. NLRC5 expression after siRNA knockdown, antigen-specific T cell-mediated killing, tumour growth in animal models, and histopathological assessment of immune cell infiltration were analysed using Student\u0026rsquo;s t-test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all analyses. All data were reproduced with at least two independent experiments.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDownregulation of\u003c/b\u003e \u003cb\u003eNLRC5\u003c/b\u003e \u003cb\u003eexpression is associated with decreased MHC-I expression in NPC tumours\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGene expression analysis of the NPC dataset (GSE68799) revealed that genes within the APM signature, as defined by Thompson and colleagues (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), were collectively expressed at lower levels in NPC tumours compared to non-NPC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Among the 17 APM genes, \u003cem\u003eNLRC5, CIITA, PSMB10, PSME1\u003c/em\u003e and \u003cem\u003ePSMB9\u003c/em\u003e were significantly downregulated in NPC. Specifically, \u003cem\u003eNLRC5\u003c/em\u003e showed the most pronounced reduction at 2.2-fold. Given that \u003cem\u003eNLRC5\u003c/em\u003e functions as a master transcriptional regulator of MHC class I genes, its expression showed a strong positive correlation with the expression of \u003cem\u003eHLA-A, HLA-B\u003c/em\u003e, and \u003cem\u003eHLA-C\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In comparison, \u003cem\u003ePSMB10\u003c/em\u003e and \u003cem\u003ePSMB9\u003c/em\u003e exhibited moderate positive correlation, while \u003cem\u003eCIITA\u003c/em\u003e and \u003cem\u003ePSME1\u003c/em\u003e showed only weak correlation with the expression of \u003cem\u003eHLA-A, HLA-B\u003c/em\u003e, and \u003cem\u003eHLA-C\u003c/em\u003e (sFig. 1a). Tumours with low \u003cem\u003eNLRC5\u003c/em\u003e expression consistently exhibited low \u003cem\u003eHLAs\u003c/em\u003e expression, indicating that \u003cem\u003eNLRC5\u003c/em\u003e downregulation may lead to impaired transcriptional activation of the MHC-I group of genes, potentially decreasing MHC-I expression in NPC. Furthermore, analysis of the KEGG antigen processing and presentation pathway in the GSE68799 dataset revealed that approximately 60\u0026ndash;70% of NPC cases exhibit downregulation of \u003cem\u003eHLA\u003c/em\u003es (sFig. 1b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMHC-I downregulated tumours overexpress MAGED4B and FJX1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo validate the DEG findings indicating MHC-I downregulation, we assessed MHC-I and MHC-II protein levels by IHC in an independent cohort of 35 NPC and 5 non-NPC tissues. The demographic and clinical characteristics of NPC patients are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In non-NPC tissues, MHC-I expression was observed in normal epithelial cells at levels that are comparable or higher than those in the surrounding stroma. By contrast, NPC tumour cells exhibited weaker MHC-I membrane staining compared to lymphocytes and endothelial cells in the surrounding stroma, indicating MHC-I downregulation in tumours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). MHC-I downregulation was detected in 63% (22/35) of NPC cases, and none in the non-NPC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The IHC findings are consistent with our gene expression analysis, confirming that MHC-I is significantly downregulated in about two-thirds of NPC cases. However, MHC-I downregulation was not associated with disease stage or lymph nodes involvement (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Meanwhile, MHC-II downregulation showed minimal differences between NPC and non-NPC tissues (29% vs 40%). Given the higher prevalence of MHC-I downregulation in NPC, further investigation into its expression and modulation was conducted in this study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the relationship between TAAs expression and MHC-I status, the same set of tissues underwent IHC staining for MAGED4B and FJX1. Both TAAs were found to be overexpressed in 91% of NPC tissues (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; sFig. 2a), which is in line with previous study (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, about 60% of NPC cases with high TAAs expression exhibited MHC-I downregulation, indicating a need for strategies to restore antigen presentation to enable effective immune surveillance and promote antigen-specific cytotoxicity (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic and clinical characteristics of NPC patients.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePatients with NPC, N (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNPC tissues\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35 (100%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWhole tissue section\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 (34.1%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21 (65.9%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRange, years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28\u0026ndash;84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;14.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32 (91.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (8.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStaging\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage I-II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22 (62.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage III-IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11 (31.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLymph nodes metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20 (57.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (37.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe association between MHC-I and MHC-II with clinical variables.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMHC-I\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eMHC-II\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ep\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003ep\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNPC tumours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22 (62.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (37.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0289*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10 (28.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e25 (71.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.6266\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNon-NPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0 (0.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4 (100.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2 (40.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3 (60.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStaging\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage I-II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14 (40.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8 (22.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7136\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5 (14.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e17 (48.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.4376\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage III-IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6 (17.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5 (14.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4 (11.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e7 (20.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLymph nodes metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13 (37.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7 (20.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3 (8.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e17 (48.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.1067\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7 (20.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6 (17.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6 (17.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e7 (20.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe association between MHC-I and MHC-II expression with clinical variables was examined with the Fisher\u0026rsquo;s exact test. *A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered to be statistically significant.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003ea\u003c/b\u003e: The association between target antigens expression in NPC tumours and non-NPC tissues.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMAGED4B\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eFJX1\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ep\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003ep\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNPC tumours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3 (8.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32 (91.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3 (8.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e31 (91.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.0005*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNon-NPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5 (100.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0 (0.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4 (100.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0 (0.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe association between MAGED4B and FJX1 expression with sample type was examined with the Fisher\u0026rsquo;s exact test. *A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered to be statistically significant.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eb\u003c/b\u003e: TAAs expression and MHC-I status in NPC tissues.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eProtein expression\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMAGED4B\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;35)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eFJX1\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eLow\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMHC-I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1 (2.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21 (60%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2 (5.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e20 (58.8%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2 (5.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (31.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1 (2.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11 (32.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide-trained T cells enhance MHC-I expression in NPC cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effect of peptide-trained T cells on MHC-I expression in NPC cells, we first screened a panel of NPC cell lines for baseline expression of MAGED4B, FJX1 and MHC-I. Both MAGED4B and FJX1 were detected across all NPC cell lines (sFig. 3a). Flow cytometry analysis revealed that NPC-268 exhibited the lowest surface MHC-I expression, followed by NPC-43, and NPC-53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). All cell lines showed MHC-I induction upon IFNg stimulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells specific for MAGED4B (M-T cells,) and FJX1 (F-T cells) were generated from healthy donors (HD23, HD30 and HD32) carrying at least one of these HLA haplotypes: HLA-A2, HLA-A11 or HLA-A24 (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These peptide-trained T cells were co-cultured with HLA-A matched NPC cell lines (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), resulting in an approximately two-fold increase in MHC-I expression in NPC cells relative to baseline levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eEpigenetic modulation such as DNA methylation and histone deacetylation (HDAC) is known to contribute to MHC-I downregulation in tumours. As the NPC cell lines used in this study were hypomethylated, DNA methylation was considered an unlikely mechanism of MHC-I suppression (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Although histone deacetylation status of these cell lines was not fully characterized, HDAC inhibition was performed as an indirect approach to probe HDAC regulation of MHC-I in these cell lines. SAHA was chosen as the HDAC inhibitor in this study because it is currently being tested in combination with ICI for recurrent and metastatic head and neck cancer (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The response to SAHA treatment varied across NPC cell lines. NPC-43 exhibited a dose-dependent increase in total and surface MHC-I expression, indicating the potential of HDAC in mediating MHC-I expression in this cell. Meanwhile, NPC-53 and NPC-268 showed minimal changes in surface MHC-I, with relative fold change below 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In comparison to SAHA treatment, peptide-trained T cells induced MHC-I upregulation across all three NPC cell lines, suggesting that a vaccine-based approach may offer a more robust strategy for enhancing MHC-I expression in these NPC cell lines.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeptide-trained T cells upregulate MHC-I-associated genes and promote antigen-specific killing of NPC cell lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the molecular basis of MHC-I upregulation in NPC by peptide-trained T cells, we analysed the expression of key genes involved in MHC-I transcription and assembly including \u003cem\u003eNLRC5\u003c/em\u003e, \u003cem\u003eTAP1\u003c/em\u003e, and \u003cem\u003eB2M\u003c/em\u003e following co-culture. Given that IFNγ stimulation is known to concurrently induce both MHC-I and CD274 (PD-L1), which can have opposing effects on antitumor immunity (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), we also examined CD274 expression in response to peptide-trained T cells.\u003c/p\u003e\u003cp\u003eCompared to control cells, \u003cem\u003eNLRC5, TAP1\u003c/em\u003e and \u003cem\u003eB2M\u003c/em\u003e mRNA levels were significantly upregulated in NPC cells following co-culture with peptide-trained T cells, although the magnitude of induction varied across cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Notably, CD274 expression was substantially lower following co-culture with peptide-trained T cells compared to direct IFNγ stimulation. These findings indicate that peptide-trained T cells preferentially enhance MHC-I while limiting excessive PD-L1 upregulation, which may help to overcome immune suppression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImportantly, antigen-specific T cells killing was observed across all three NPC cell lines following MHC-I upregulation induced by peptide-trained T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After 24 hours of co-culture, these peptide-trained T cells achieved cytolytic efficiencies ranging from 50\u0026ndash;60%, exceeding the 20% baseline for non-specific killing, regardless of the differences in basal MHC-I expression in these cell lines. For example, NPC-268, which initially exhibited low MHC-I levels, showed enhanced MHC-I expression and demonstrated susceptibility to T cell-mediated cytotoxicity, achieving a similar cytolysis rate compared to NPC-43 and NPC-53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The induction of MHC-I potentially suggests improved tumour cell recognition that leads to antigen-specific cytolysis. Furthermore, CD8⁺ T cells derived from multiple donors consistently demonstrated antigen-specific killing, confirming the reproducibility of this response across individuals (sFig3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eNLRC5\u003c/b\u003e \u003cb\u003eknockdown reduces MHC-I expression in NPC cells without impairing T cell-mediated cytotoxicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eNLRC5\u003c/em\u003e is an important regulator of MHC-I expression. To assess the effect of \u003cem\u003eNLRC5\u003c/em\u003e on MHC-I expression and T cell-mediated cytotoxic efficiency, we used siRNA to knockdown \u003cem\u003eNLRC5\u003c/em\u003e in NPC-43. \u003cem\u003eNLRC5\u003c/em\u003e knockdown in NPC-43 cells was validated at the gene level, showing a 76% reduction in NLRC5 mRNA expression, which remained low for up to 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). This knockdown was further confirmed at the protein level, which also resulted in a decrease in both NLRC5 and MHC-I expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Interestingly, while NLRC5 knockdown markedly reduced MHC-I expression, it did not affect the expression of other key MHC-I associated genes such as \u003cem\u003eB2M\u003c/em\u003e and \u003cem\u003eCD274\u003c/em\u003e, but led to an increase in \u003cem\u003eTAP1\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDespite reduced MHC-I expression following NLRC5 knockdown, no significant difference in tumour cell killing between control and siNLRC5-treated cells was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). This is likely due to the restoration of NLRC5 and MHC-I expression upon co-culture with peptide-trained T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These findings suggested that vaccine can upregulate both NLRC5 and MHC-I expressions thereby enhancing target cell susceptibility to T cell-mediated cytotoxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVaccine-induced upregulation of\u003c/b\u003e \u003cb\u003eNLRC5\u003c/b\u003e \u003cb\u003eenhances antigen presentation and suppresses tumour growth\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDue to the lack of syngeneic NPC mouse model, we employed the B16F10-A2 mouse melanoma characterized by poor immunogenicity and low MHC-I expression to evaluate the efficacy of the DNA vaccine in an antigen presentation-deficient setting (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Although limited to a single model, our \u003cem\u003ein vivo\u003c/em\u003e efficacy study demonstrated that a prime-boost regime of the DNA vaccine significantly suppressed tumour growth compared to the pLDOM control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Vaccine-treated tumours also demonstrated a significant increase in CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration as compared to control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), suggesting enhanced antitumor immunity in poorly immunogenic B16F10 tumours.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate whether vaccine modulates antigen presentation, we performed RNA sequencing on the tumour samples. Our KEGG pathway enrichment analysis identified antigen processing and presentation as one of the most significantly upregulated pathways following DNA vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). This was supported by the overall upregulation of genes of the APM signature. In particular, \u003cem\u003eNlrc5\u003c/em\u003e, \u003cem\u003eTap1\u003c/em\u003e and \u003cem\u003eRfx5\u003c/em\u003e were the genes significantly increased in the DNA vaccine-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The \u003cem\u003ein vivo\u003c/em\u003e findings align with our \u003cem\u003ein vitro\u003c/em\u003e data, where co-culture of NPC cells with peptide-trained T cells upregulated NLRC5, TAP1, and B2M, restored MHC-I expression, and enhanced antigen-specific cytotoxicity. Together, these results demonstrate that the vaccine can upregulate NLRC5 and promote antigen presentation, thereby strengthening anti-tumour immune responses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancers can evade immune surveillance by downregulating APM, which in turn impairs antigen presentation and reduces immunogenicity. In about 30% of NPC cases with APM downregulation, somatic alterations such as mutations and structural variants affecting MHC class I expression were found in NLRC5, HLA-A, and HLA-B, occurring in 2.9 to 8.5 percent of cases (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). This observation suggested that only a small subset of APM-deficient tumours may represent hard lesions with irreversible genetic changes, while others may be soft lesions with potentially reversible suppression.\u003c/p\u003e\u003cp\u003eIn this study, we identified that APM downregulation in NPC may arise from the transcriptional downregulation of \u003cem\u003eNLRC5\u003c/em\u003e, a key transcriptional regulator of MHC-I expression (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Indeed, we showed that NLRC5 expression is strongly correlated with MHC-I expression in NPC. NLRC5 is critical for mediating MHC-I-dependent CD8\u003csup\u003e+\u003c/sup\u003e T cell antitumor immunity (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Studies have shown that NPC patients with MHC-I downregulation exhibit reduced CD8\u003csup\u003e+\u003c/sup\u003e T cell populations in both tumours and peripheral blood compared to healthy controls, indicating impaired CD8\u003csup\u003e+\u003c/sup\u003e T cell-mediated antitumor immunity (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Furthermore, low NLRC5 expression is associated with poor patient survival, underscoring its clinical relevance (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMAGED4B and FJX1 are shared TAAs and were found to be upregulated in 91% of NPC cases, consistent with the 95% reported in head and neck cancer (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). These TAAs contribute to oncogenesis by promoting cancer cell proliferation and invasion (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Importantly, MAGED4B and FJX1 are reported to be immunogenic, and their high expression in NPC highlights their potential as viable targets for vaccine-based therapies. However, 60% of NPC cases overexpressing these TAAs exhibit downregulation of MHC-I. This paradox underscores a critical therapeutic opportunity: although TAAs are abundantly expressed, their recognition by cytotoxic T cells is compromised. Therefore, restoring MHC-I-mediated antigen presentation is essential to enhance immune recognition. Here, we demonstrate that a cancer vaccine approach can effectively restore antigen presentation and enhance immunogenicity in APM-downregulated NPC.\u003c/p\u003e\u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e experiments demonstrated that co-culturing peptide-trained T cells with NPC cell lines induced the expression of NLRC5 and MHC-I across all three NPC cell lines, along with key MHC-I-associated genes, TAP\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eB2M\u003c/em\u003e. While a potent induction of NLRC5 could be achieved through IFNγ stimulation, the clinical use of systemic IFNγ is limited due to its association with severe and potentially life-threatening toxicities (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Alternatively, the FDA-approved HDAC inhibitor SAHA has been shown to enhance MHC-I expression by reversing epigenetic silencing in various cancer models (\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). However, in our study, SAHA exhibited limited efficacy in the NPC cells. Only NPC-43 showed dose-dependent induction of MHC-I, while NPC-53 and NPC-268 displayed minimal response even at the highest concentration tested. Although HDAC status in NPC cell lines is not known, we postulated that HDAC-mediated epigenetic suppression may not be the underlying mechanism of MHC-I downregulation in these NPC cell lines tested.\u003c/p\u003e\u003cp\u003eThis upregulation of NLRC5 and MHC-I protein expression by peptide-trained T cells enhanced tumour immunogenicity and promoted antigen-specific cytotoxicity against NPC cell lines. To further investigate the role of NLRC5, we performed knockdown experiments in NPC cells. Although NLRC5 deficiency led to reduced MHC-I expression, it was reversed upon co-culture with peptide-trained T cells, resulting in T cell-mediated cytotoxicity. Consistently, our \u003cem\u003ein vivo\u003c/em\u003e studies showed that antigen-specific DNA vaccination enhances antitumor immunity in a MHC-I downregulated antigen-expressing B16F10-A2 melanoma syngeneic mouse model. Notably, tumour suppression was accompanied by increased CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration following vaccination. Consistent with our \u003cem\u003ein vitro\u003c/em\u003e study, the APM pathway was among the most significantly upregulated pathways in DNA vaccine-treated tumours. Among the 17-gene APM signature, \u003cem\u003eNlrc5\u003c/em\u003e was found to be significantly upregulated in mice responding to DNA vaccination. The induction and stable expression of \u003cem\u003eNlrc5\u003c/em\u003e could be crucial for MHC-I upregulation, which further restores immunogenicity in B16F10 melanoma cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Hence, we postulated that peptide-trained T cells naturally secrete inflammatory cytokines such as IFNγ and TNFα, which activate JAK-STAT signalling to induce NLRC5 transcription (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). NLRC5, in turn, binds to promoter regions to enhance the transcription of MHC-I, B2M, and TAP1, thereby promoting antigen presentation (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Additionally, others have shown that NLRC5 can also regulate IFNγ response, providing a feedback loop between NLRC5 and IFNγ that sustains MHC-I upregulation (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStrategies aimed at inducing IFNγ could potentially enhance APM expression. However, it is important to consider that while IFNγ is crucial for initiating immune responses, excessive IFNγ stimulation may induce CD274 in the absence of specific antigen recognition and lead to T cell dysfunction (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Therefore, approaches that rely on antigen-specific activation, such as cancer vaccines, may offer a more targeted and clinically feasible strategy for restoring antigen presentation in NPC. Moreover, it is also worth noting that MHC-I expression can be regulated through alternative pathways, such as NF-κB and cGAS-STING signaling (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), which were not examined in this study. Nevertheless, our findings highlight the potential of vaccination strategies to counteract transcriptional suppression of APM genes and overcome immune evasion mechanisms in NPC.\u003c/p\u003e\u003cp\u003eNPC is characterized by abundant immune cell infiltration and high CD274, yet its response rate to ICIs remains limited compared to other \"hot\" tumours. One contributing factor is the downregulation of APM, including MHC-I (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Restoring MHC-I expression has been shown to improve tumour sensitivity to ICI treatment (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). In our study, peptide-trained T cells robustly induced MHC-I-associated components in NPC cells, and only minimally upregulated CD274. This is notable because PD-L1 expression is often co-regulated with MHC-I in response to IFNγ. The modest induction of CD274 alongside strong MHC-I upregulation suggests that vaccination may selectively enhance tumour immunogenicity without triggering excessive immune checkpoint expression.\u003c/p\u003e\u003cp\u003eOur study has several limitations that require further investigation. This study suggests that cancer vaccines may enhance immune recognition in tumours with transcriptionally suppressed MHC-I. However, MHC-I downregulation may also result from enhanced protein degradation and disruptions in endocytic trafficking, which are not explored in the current study and whose responsiveness to cancer vaccination remains unknown. Due to the lack of a syngeneic NPC mouse model, \u003cem\u003ein vivo\u003c/em\u003e experiments were performed using the B16F10 melanoma model. Although this model may not fully recapitulate the NPC tumour microenvironment, it served as a useful platform to confirm APM modulation following vaccination. These limitations underscore the need for employing more representative NPC models and comprehensive analyses of MHC-I regulatory pathways to better understand immune evasion and optimize immunotherapeutic strategies in NPC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study demonstrates that peptide-trained T cells can upregulate MHC-I expression in NPC cells via NLRC5 transcriptional regulation, enhancing immune recognition and cytotoxic killing of tumour cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These findings highlight the potential of cancer vaccines in APM-downregulated tumours, presenting a potential strategy to enhance immunotherapy efficacy, particularly for patients with MHC-I downregulation and resistance to current treatments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eNPC\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNasopharyngeal carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eICI\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmune checkpoint inhibitor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eNLRC5\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNOD-like receptor family CARD domain containing 5\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eAPM\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAntigen presentation machinery\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMHC-I\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMajor histocompatibility complex class I\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eMAGED4B\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMAGE family member 4B\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eFJX1\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFour-jointed box kinase 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eTAA\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumour-associated antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eIFNγ\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterferon gamma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eSAHA\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSuberoylanilide hydroxamic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eCIITA\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eClass II major histocompatibility complex transactivator\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eTAP1\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransporter associated with antigen processing 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBeta-2-microglobulin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Touchlight Genetics Ltd for generously providing the doggybone DNA vaccine and for their valuable input and discussions throughout the course of this project. This study also utilised NPC cell lines generously provided by Translational Cancer Biology Research Unit, Cancer Research Malaysia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Technology Development Fund 2, TeD2 (TEF 04221148) from the Ministry of Science Technology and Innovation (MOSTI), Malaysia, Touchlight Genetic Ltd, United Kingdom and internal funding from Cancer Research Malaysia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChai Phei Gan and Sau Yee Kok contributed equally to this work.\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eCancer Immunology \u0026amp; Immunotherapy Research Unit, Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChai Phei Gan \u0026amp; Sau Yee Kok \u0026amp; Natasha Zulaziz \u0026amp; Sok Ching Cheong \u0026amp; Kue Peng Lim\u003c/p\u003e\n\u003cp\u003eDepartment of Mathematical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia\u003c/p\u003e\n\u003cp\u003eBernard Kok Bang Lee\u003c/p\u003e\n\u003cp\u003eMolecular \u0026amp; Clinical Cancer Medicine, University of Liverpool, UK\u003c/p\u003e\n\u003cp\u003eNatalia Savelveya\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eConception and design, CPG, SYK SCC, and KPL; Experiment performance; CPG, SYK, NZ, and KPL; Data analysis and interpretation, CPG, SYK, BKBL, and LKP; Manuscript preparation, CPG, SYK, BKBL, and KPL. Manuscript revision, CPG, SYK, BKBL, NZ, SCC, NS and KPL. All authors contributed, studied, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eKue Peng Lim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis work was approved by\u0026nbsp;the Medical Ethics Committee of Penang General Hospital (NMRR-09-944-4848) and Faculty of Dentistry, University of Malaya [DF OS 2101/0002/2179(L). Informed consent was obtained from all donors. The animal study was approved by Experimental Animal Ethics Committee, National University Malaysia (CRM/2020/KUE PENG/25-MAR./1093-APR.-2020-APR.-2023).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors approved the contents of the manuscript and agreed with the publication.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eKPL and NS received funding from Touchlight Genetics Ltd for works in the development of cancer vaccine. The other authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePublicly available data used in this study were obtained from the NCBI Gene Expression Omnibus (GEO) under accession number GSE68799. The RNA-seq data generated in this study are not publicly available due to ongoing related studies but are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCant\u0026ugrave; G. Nasopharyngeal carcinoma. A different head and neck tumour. Part A: from histology to staging. Acta Otorhinolaryngol Ital. 2023;43:85\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee AWM, Ng WT, Chan LLK, Hung WM, Chan CCC, Sze HCK, et al. Evolution of treatment for nasopharyngeal cancer \u0026ndash; Success and setback in the intensity-modulated radiotherapy era. Radiother Oncol. 2014;110:377\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerri F, Della Vittoria Scarpati G, Caponigro F, Ionna F, Longo F, Buonopane S, et al. Management of recurrent nasopharyngeal carcinoma: current perspectives. 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Int J Mol Sci. 2021;22(13):6741.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu B, Ouda R, An N, Tanaka T, Kobayashi KS. The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation. J Biol Chem. 2024;300(5):107205.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu SS, Zhang W, Wang X, Jiang P, Traugh N, Li Z, et al. Therapeutically Increasing MHC-I Expression Potentiates Immune Checkpoint Blockade. Cancer Discov. 2021;11(6):1524\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin Q, Zhou Y, Ma J, Han S, Huang Y, Wu F, et al. Single-cell analysis reveals the multiple patterns of immune escape in the nasopharyngeal carcinoma microenvironment. Clin Transl Med. 2023;13:e1315.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang B, Li J, Hua Q, Wang H, Xu G, Chen J, et al. Tumor CEMIP drives immune evasion of colorectal cancer via MHC-I internalization and degradation. J Immunother Cancer. 2023;11(1):e005592.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nasopharyngeal Cancer, Cancer Vaccine, Tumour Associated Antigen, Antigen Presentation Machinery, Immunogenicity, T Cell Cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-7037136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7037136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNasopharyngeal carcinoma (NPC) is considered an immune-hot tumour. However, 30\u0026ndash;80% of cases exhibit downregulation of antigen processing and presentation machinery (APM), enabling the evasion of host immunosurveillance. While cancer vaccines aim to trigger immune responses against tumour antigens, their efficacy in APM-deficient tumours remains uncertain. This study evaluates the efficacy of cancer vaccine targeting tumour-associated antigens in APM-downregulated NPC cells \u003cem\u003ein vitro\u003c/em\u003e and further validates the modulation of APM pathways \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eAPM status was evaluated via differential gene expression analysis of 42 NPC tumours and 4 non-NPC tissues using a 17-gene APM signature. Genes downregulated in NPC and associated with reduced MHC-I expression were identified. MHC-I expression was further examined by immunohistochemistry in 35 tumours and 5 non-NPC tissues. The effect of cancer vaccine on APM gene expression was examined by co-culturing peptide-trained T cells with NPC cells. Next, T cell-mediated cytotoxicity was assessed in an APM-deficient model generated by siRNA-mediated knockdown of \u003cem\u003eNLRC5\u003c/em\u003e. Finally, cancer vaccine-induced modulation of APM genes was validated in a poorly immunogenic mouse tumour model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAmong the 17-gene APM signature, \u003cem\u003eNLRC5\u003c/em\u003e was the most significantly downregulated gene in NPC and strongly correlated with reduced MHC-I expression. Immunohistochemistry confirmed MHC-I downregulation in 63% of tumours. Co-culture of NPC cells with peptide-trained T cells upregulated NLRC5, and key MHC-I assembly genes (TAP1 and B2M), enhancing MHC-I expression and antigen-specific cytotoxicity in NPC cells expressing the target antigen. Notably, the knockdown of NLRC5 was reversed upon co-culture with peptide-trained T cells, resulting in T cell-mediated cytotoxicity. \u003cem\u003eIn vivo\u003c/em\u003e, cancer vaccine treatment consistently induced APM gene expression, including NLRC5, supporting its potential in restoring antigen presentation in NPC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study demonstrates that peptide-trained T cells can upregulate NLRC5 and MHC-I expressions on tumour cells, thereby restoring antigen presentation and enhancing tumour immunogenicity. These findings underscore the therapeutic potential of cancer vaccines in treating APM-downregulated NPC.\u003c/p\u003e","manuscriptTitle":"Cancer vaccine overcomes immune evasion of nasopharyngeal carcinoma by restoring MHC-I through transcriptional regulation of NLRC5","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 08:06:47","doi":"10.21203/rs.3.rs-7037136/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-14T16:48:30+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T16:17:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-05T16:18:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-07-03T05:30:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f36d1642-4870-492a-b43b-5a54e888f17c","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:03:33+00:00","versionOfRecord":{"articleIdentity":"rs-7037136","link":"https://doi.org/10.1186/s12967-025-07418-x","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-12-23 15:58:34","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-07-22 08:06:47","video":"","vorDoi":"10.1186/s12967-025-07418-x","vorDoiUrl":"https://doi.org/10.1186/s12967-025-07418-x","workflowStages":[]},"version":"v1","identity":"rs-7037136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7037136","identity":"rs-7037136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}