In all, 761 patients with newly diagnosed DLBCL were included in this study. Two experienced pathologists (YHM and WCF) established histological diagnosis according to the World Health Organization classification. All patients were treated with R-CHOP-based immunochemotherapy. The treatment response was evaluated according to the International Workshop Criteria. The Hospital Review Board approved the study with informed consent obtained following the Declaration of Helsinki.
Cell lines and reagents
B-lymphoma cell line SU-DHL-4 (obtained from American Type Culture Collection, Manassas, VA, USA) were grown in Roswell Park Memorial Institute (RPMI)-1640 medium and OCI-LY7 (kindly provided by HCX) were grown in Iscove's Modified Dulbecco's Medium (IMDM), supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (15140122, Gibco, Carlsbad, CA, USA) in a humidified atmosphere containing 95% air-5% CO2 at 37°C. γ-secretase inhibitor (GSI-I) was purchased from Selleck (Houston, TX, USA).
Tumor samples of 761 patients were analyzed for gene mutations using WGS/WES or targeted sequencing. WGS (n=109) was performed on frozen tumor tissue. WES (n=225) was performed on frozen tumor tissue and formalin-fixed paraffin-embedded tumor tissue quality-controlled by agarose gel electrophoresis. Targeted sequencing (n=427) was performed on frozen tumor tissue. Mutation frequencies per gene and mutation signatures showed no significant difference in the results for WGS, WES and targeted sequencing.
Total RNA was extracted from tumor samples of 402 DLBCL patients. RNA was purified using Ribo-Zero rRNA Removal Kits (Illumina). RNA concentration and integrity were verified using NanDrop and Agilen 2100 Bioanalyzer, respectively. RNAs libraries were constructed with TruSeq RNA Library Preparation Kit (Illumina). The concentration and the quality of libraries were controlled by Qubit and BioAnalyzer 2100 system. The paired-end sequencing was performed on Illumina HiSeq Sequencer. After 3' adaptor-trimming and removing low-quality reads, high quality trimmed reads were aligned to the reference genome (UCSC hg19). Pathway enrichment analysis was performed based on the differentially expressed mRNAs through the user tutorials of Cytoscape referring to Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Gene Set Enrichment Analysis (GSEA) was performed using the BROAD Institute GSEA software (http://www.broad.mit.edu/gsea/).
For Vector, KMT2DR5432Q, KMT2Dwt, KMT2Dkd and Scramble transfection, purified plasmids pGV358/GFP/Puro (Vector), pGV358/GFP/Puro-KMT2D (NM-003482, residues 4839-5537, containing SET domain, wild-type, wt), pGV358/GFP/Puro-KMT2D (NM-003482, residues 4839-5537, containing SET domain, R5432Q), pGV248/GFP/Puro (Scramble), pGV248/GFP/Puro-sh KMT2D were transfected into packages HEK-293T cells using lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The supernatant fraction of HEK-293T cell cultures was then condensed to a viral concentration of approximately 2 × 108 transducing units/ml. The lentiviral particles were incubated with SU-DHL-4 or OCI-LY7 cells for 72h with addition of polybrene (8μg/ml). The stably transduced clones were selected by green and/or red fluorescence protein using flow cytometry or puromycin treatment for two weeks. The shRNA sequences of KMT2D were listed in Table. S1
Cells were lysed in 200μl lysis buffer (0.5M Tris-HCl, pH 6.8, 2mM EDTA, 10% glycerol, 2% SDS and 5% β-mercaptoethanol). Protein lysates (20μg) were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk and incubated overnight at 4°C with appropriate antibodies, followed by a horseradish peroxidase-conjugated secondary antibody. The immunocomplexes were visualized using a chemiluminescence phototope-horseradish peroxidase Kit (Cell Signaling Technologies, Danvers, MA, USA). Primary antibodies included H3K4me3 (ab8580, Abcam, Cambridge, United Kingdom), FBXW7 (ab109617, Abcam), NICD (ab8925, Abcam), MYC (ab32072, Abcam), TGF-β1 (ab215715, Abcam). Histone 3 (17168, Proteintech) and β-Actin (ab8226, Abcam) were used to ensure equivalent loading of protein. Horseradish peroxidase-conjugated secondary antibodies against goat anti-mouse-IgG and goat anti-rabbit-IgG were from Cell Signaling Technologies.
Immunohistochemistry and immunofluorescence assay
Immunohistochemistry was performed on 5μm-paraffin sections with an indirect immunoperoxidase method using antibodies against MYC (1:500, ab32072, Abcam), H3K4me3 (1:1000, ab8580, Abcam), NICD (1:200, ab8925, Abcam) and Foxp3 (1:500, ab20034, Abcam). H3K4me3 and NICD expression levels were scored based on the percentage of positive cells: +, < 25%; ++, 25-49%; +++, 50-74%; ++++, 75-100%. For Foxp3, immunoreactivity in > 0% of cells was defined as positive . Immunofluorescence assay of H3K4me3 was performed on acetone-fixed cells, using rabbit anti-H3K4me3 as primary antibodies and Alexa Fluor 594-conjugated goat polyclonal anti-rabbit IgG-H&L (ab150080, Abcam) as the secondary antibody. Nuclei were counterstained with DAPI.
Quantitative real-time PCR (RT-PCR)
Total mRNA was extracted using Trizol reagent and reverse transcribed using a PrimeScript RT Reagent Kit with gDNA Eraser for quantitative RT-PCR (RR047A, TaKaRa, Japan). Quantitative RT-PCR was performed by SYBR Premix Ex TaqTM II (RR820A, TaKaRa) and ABI ViiA7 (Applied Biosystems, Bedford, MA, USA) with primers against TGF-β1, FBXW7 and MYC. Relative quantification was calculated using the 2-∆∆CT methods. The primers were listed in Table. S2.
Chromatin immunoprecipitation (ChIP)
Nuclear extracts were prepared from 2 × 107 cells per sample. Rabbit anti-human H3K4me3 antibody (ab8580, Abcam) was used for immunoprecipitation, and normal IgG (3900, Cell Signaling Technologies) was referred as negative control. ChIP primers of FBXW7 genes were used as previously reported , which were designed to detect promoter fragments near transcription start sites. ChIP-enriched chromatin was used for real-time PCR with SYBR Premix Ex TaqTM II, normalizing to input.
B-lymphoma cell co-culture
Co-culture of SU-DHL-4 or OCI-LY7 cells with PBMCs was conducted by a 0.4μm pore polycarbonate membrane and 6.5mm inserts (Corning, Sunnyvale, CA, USA). SU-DHL-4 and OCI-LY7 cells transfected with Vector, KMT2Dwt, KMT2DR5432Q, Scramble, and KMT2Dkd were cultured at 2 × 105 cells/ml in the upper chamber, while PBMCs at 1 × 106 cells/ml (1:5 ratio) in the lower chamber. Cell viability was assessed by CCK8. After 72h co-culture, we measured its absorbance at 450nm by spectrophotometry. All cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin.
Single-cell RNA sequencing
Tumor samples of six DLBCL patients were collected for single-cell RNA sequencing analysis. Tumor samples were placed in separate containers containing Tissue Storage Solution (Miltenyi Biotec) and transported in regular ice to the laboratory immediately. Tumor samples were separated into small pieces mechanically and dissociated into single-cells after enzymatic digestion at 37 ℃. After filtering with a 40-µm strainer and washing once with PBS, cells were cryopreserved in liquid nitrogen before the scRNA-seq experiment. For the scRNA-seq experiment, cells were thawed and washed immediately, and further processed with Dead cell removal kit (Miltenyi Biotec). The preparation of the single-cell suspensions, synthesis of complementary DNA and gene expression libraries were performed according to the manufacturer’s instructions using Chromium single cell 3’ Kit v2 (10x Genomics) in 2 patients, and Chromium single cell 5’ Kit v2 (10x Genomics) in 4 patients. The 3’ gene expression libraries were sequenced on Novaseq 6000 (Illumina) and 5’ gene expression libraries on MGI-2000 sequencer.
Reading the raw data by the Read10X function in the seurat package (4.0.2)  and constructed the analysis data by the CreateSeuratObject function. The data filtering criteria were: minimum number of cells was 3, minimum number of genes tested was 300, and the percentage of mitochondrial gene expression was less than 5%. The data were first normalized by LogNormalize method, and the top 1500 genes with large intercellular variation coefficients were extracted by FindVariableFeatures function. The normalized count matrix is linearly transformed ("scaling") by the ScaleData function to normalize the data: 1) shifting the expression of each gene so that the average expression between cells is 0; 2) scaling the expression of each gene so that the difference between cells to 1. The normalized expression matrix was subjected to PCA downscaling using the RunPCA function, and only the 1500 highly variable genes selected earlier were linearly downscaled.
The distribution of p-values for each PC was calculated using the JackStraw function. Significant PCs would have lower p-values, and the appropriate PC values were filtered. The above descended data were clustered by t-distributed stochastic neighbor embedding (tSNE) algorithm, and the differential gene analysis was performed by FindAllMarkers function for each subpopulation to filter Marker genes. The subpopulations were annotated according to marker genes by SingleR package (1.4.1) , in which the annotation comparison data set was HumanPrimaryCellAtlasData, and the similarities and differences of cell numbers between cancer and normal groups were counted after subpopulation annotation. The expression matrices of T cells in the cancer group and normal group were extracted separately, and the data were constructed separately by CreateSeuratObject function, then the two groups of data were merged by merge function. The differential expression genes of T cells in the cancer group and normal group were calculated by FindAllMarkers function. The screening criteria for differentially expressed genes were P value < 0.05.
Antibodies used for cell labeling of Treg cells were as followed: BV421 anti-CD4 (562424, BD Biosciences), FITC anti-CD3 (317306, Biolegend), BV786 anti-CD25 (563701, BD Biosciences), PE anti-Foxp3 (560046, BD Biosciences). Flow cytometry data were collected by a FACS Calibur cytometer (BD Biosciences) and analyzed by FlowJo software.
Luciferase report assay
Total cDNA from HEK-293T cells was used to amplify the promoter (-1914 to +79 bp) of TGF-β1, forward primer: 5′-GCCCGCAACATATAGATGAGGACGGTGGCCCAGCCC-3′; reverse primer: 5′-CTATATGTTGCGGGCTCCGAGGGGGGTC-3′. After digestion with BamHI and EcoRI, PCR products were ligated into the GM-4629：PGL3-basic vector and confirmed by DNA sequencing. HEK-293T cells were seeded in 24-well plates and co-transfected with 250ng of MYC, 250ng promoter (-1914 to +79 bp) luciferase reporter construct and 50μl luciferase reporter. Cells were collected 24h after transfection, using the Cell Lysis Buffer (100μl per well) provided as part of the Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI, USA). Firefly and Renilla luciferase activities were examined by the Dual-Luciferase Reporter Assay System and detected by a Centro XS3 LB960 Luminometer (Berthold).
NOD-PrkdcscidIl2rgem1/Smoc (M-NSG) female mice were used in this study, which purchased from Shanghai Model Organisms, ages 6 to 8 weeks at experiment initiation. M-NSG mice were injected s.c. with KMT2Dwt and KMT2DR5432Q SU-DHL-4 cells (1 × 107 cells) and human PBMC i.v. (5 × 106 cells). Tumor volumes were measured twice a week and human CD45 (hCD45) were measured after 3 weeks to confirm that humanized PBMCs were established. TGF-β inhibitor (purchased from Selleck, SB431542) was injected i.p. in KMT2DR5432Q mice at a dose of 10mg/kg per week, 7 days after tumor cell injection. Tumor volumes were calculated at 0.5 × a (length) × b (width)2. Animals were used according to the protocols approved by the Shanghai Rui Jin Hospital Animal Care and Use Committee.
The baseline characteristics of patients were measured by χ2 test. Progression-free survival (PFS) was calculated from the date when treatment began to the date when the disease progression was recognized or the date of last follow-up. Overall survival (OS) was measured from the date of diagnosis to the date of death or last follow-up. Univariate hazard estimates were generated with unadjusted Cox proportional hazards models. Survival functions were estimated using the Kaplan-Meier method and compared by the log-rank test. Experimental results were calculated as the mean ± standard deviation from three separate experiments. The student t-test was applied to compare two normally distributed groups and Mann-Whitney U test to compare which did not conform to normal distribution. All statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS, 26.0) software or GraphPad Prism 8 software. Statistical significance was defined as p < 0.05.