Blue indicates decreased expression and red indicates increased expression compared to the IL-21 untreated control. and is being explored as a new therapeutic strategy for this type of lymphomas. However, our previous studies showed Cefpiramide sodium that IL-21 stimulation of EBV-positive DLBCL cell lines leads to increased proliferation. Here, analysis of a rare clinical sample of EBV-positive DLBCL, in combination with a NOD/SCID mouse xenograft model, confirmed the effect of IL-21 on the proliferation of EBV-positive DLBCL cells. Using RNA-sequencing, we identified the pattern of differentially-expressed genes following IL-21 treatment and verified the expression of key genes at the protein level using western blotting. We found that IL-21 upregulates expression of the host and AP-1 (composed of related Jun and Fos family proteins) and STAT3 phosphorylation, as well as expression of the viral LMP-1 protein. These proteins are known to promote the G1/S phase transition to accelerate cell cycle progression. Furthermore, in NOD/SCID mouse xenograft model experiments, we found that IL-21 treatment increases glucose uptake and angiogenesis in EBV-positive DLBCL tumours. Although more samples are needed to validate these observations, our study reconfirms the adverse effects of IL-21 on EBV-positive DLBCL, which has implications for the drug development of DLBCL. and AP-1 in EBV-positive DLBCL. Western blotting results of the primary cells and of the EBV-positive DLBCL cell line Farage verified the predictions at the protein expression level that IL-21 specifically upregulated c-Jun, cyclin D2, cyclin E1 expression and Rb phosphorylation. To explore the role of IL-21 in promoting the proliferation of EBV-positive DLBCL cells in vivo, we conducted a complement of experiments and evaluated the in vivo efficacy of IL-21 in EBV-positive DLBCL xenograft tumour experiments. This work successfully combined dry and wet laboratory research. In the NGS analysis, we not only combined published public data, but also produced valuable, novel NGS data for EBV-positive DLBCL (including data from a rare clinical sample). We expect that this work will contribute to future research on the role of the microenvironment in EBV-positive DLBCL and provide guidance for the proper use of IL-21 in NHL treatment. Results IL-21 promotes cell viability and survival of primary cells derived from an EBV-positive DLBCL clinical sample To confirm our previous finding on the EBV-positive DLBCL cell line Farage that IL-21 induced cell proliferation rather than apoptosis, we collected primary cells (named Patient-1) from a clinical sample of EBV-positive DLBCL. After 48?h of IL-21 treatment, we observed a significant apoptosis reduction in these cells (Fig.?1a) compared to the significantly increased apoptosis in EBV-negative DLBCL primary tumours under similar conditions as previously reported16. In addition, IL-21 promoted the viability of the primary cells and of the EBV-positive DLBCL cell line Farage, but reduced viability in the EBV-negative DLBCL cell line MC116 (Fig.?1b). The total cell number of EBV-positive DLBCL cells increased significantly after 48?h with/without IL-21 treatment, indicating cell proliferation Cefpiramide sodium in both cases. Using RNA-seq analysis of EBV latency gene transcripts, we found that the EBV-positive DLBCL primary cells expressed the full set of EBV latency genes (indicating a type III latency), which is similar to Farage cells (Fig.?1d). and served as the house-keeping genes2. After IL-21 treatment, the expression of Blimp-1 that orchestrates plasma cell differentiation and the viral protein LMP-1 was upregulated in the patient-derived cells as shown by RNA-seq analysis and western blot (Fig.?1c,e), and phosphorylation of STAT3 was also upregulated (Fig.?1e). These results are the same as our previously described in the EBV-positive DLBCL cell line Farage after IL-21 treatment13,18. The RNA-seq analysis, combined with our previously reported Western blot results13,18 suggests that the expression and regulation of these key genes are similar in Farage cells and the primary cells (Fig.?1cCe), which confirmed our finding in cell lines using a primary sample. Open in a separate window Figure 1 Analysis of apoptosis, viability and gene expression of EBV-positive DLBCL cells after exposure to IL-21. (a) The primary cells derived from the EBV-positive DLBCL clinical sample (labelled Patient-1) were treated with IL-21 (100?ng/mL for 48?h) or left untreated. Samples were stained with anti-Annexin EBR2 V antibodies to measure cell apoptosis by flow cytometry. The experiment was done in Cefpiramide sodium triplicate and one representative sample is shown. Statistical analysis of the flow cytometry data was collected from 3 sample replicates. (b) The viability of EBV-positive and EBV-negative DLBCL cells was assessed by erythrosine B after 48?h of treatment with IL-21. Data in a and b are expressed as the means??SEMs (n?=?3); two-tailed t test, **P? ?0.01, *P? ?0.05. (c) The indicated DLBCL cell lysates were probed with the indicated antibodies. -Actin served as the loading control. (d) Z-score normalized log2-(FPKM?+?1) of EBV latency genes in gene expression are shown in the heatmap. and were served as the house-keeping gene control. RNA-seq data.