Figure 2.

The pivotal role of LMP1⁺ NK_C9_CXCL13 cells in oncogenic activation and development of NKTCL. A) UMAP plot showing 43713 NK cells grouped into ten clusters, including four normal clusters and six malignant clusters. Each dot represents a cell, colored according to its cell cluster as indicated at the right panel. B) Multiplex IF staining for NK_C9_CXCL13 malignant NK cells (CXCL13⁺LMP1⁺) in NKTCL biopsies from the SC‐cohort. CXCL13 and LMP1 proteins as well as nuclear DNA are detected with different colors as indicated on top. Images are representative of biological replicates from three patients. C) Heatmap showing the expression levels of selected genes (rows) for each NK cell cluster (columns). NK clusters of either normal (blue) or malignant NK (purple) are indicated as rectangles on top. Filled colors from blue to red in the squares represent normalized expression levels from low to high as scaled in row direction (row Z‐score). D) Experimental design for the in vivo blockade of LMP1 in BALB/c nude mice. For lentivirus‐mediated LMP1 knockdown (top panel), NKTCL mouse models were established with tumor initiation of NKTCL cells (YT) infected with sh‐LMP1 or control lentiviruses. For adeno‐associated virus (AAV)‐mediated LMP1 knockdown (bottom panel), NKTCL mouse models were established with tumor initiation of wild‐type YT cells, and intratumor injection of AAV‐expressing sh‐LMP1 or corresponding control was performed when the xenografts had grown to a certain volume. E) Tumor growth of xenografts derived from YT cells infected with lentivirus‐expressing shRNAs targeting LMP1 (sh‐LMP1‐1/‐2) or scrambled (sh‐Luci) at different time courses (day), with the tumor size (F) left panel) and tumor weight (F) right panel) for the excised xenografts. G) Multiplex IF staining assays (top panel) for the protein expression of Ki‐67 and c‐PARP1 in malignant NK cells for the tumor section of the xenografts excised from (E) and bar plots (bottom panel) for the percentages of Ki‐67⁺/c‐PARP1⁺ cells. H) Pseudotime development trajectories of malignant and normal NK cells. Each dot represents a cell in the trajectory projection, colored according to NK cell clusters. The inlet plot shows cells colored according to predicted pseudotime scores from deep blue to yellow, representing cell states from early stage to terminal stage, and two dashed curves represent the maturation of normal NK cells (left) and developmental process of malignant NK cells (right). Comparisons were made using Student's t‐test, and results for growth curves and bar plots are shown as mean value ± standard deviation (SD). ^ns p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 100 µm.