Fig 1: Chemotaxis regulation by malignant NK cells in NKTCL TME. A) Dot plots showing the expression of chemokine ligands (left panel) and receptors (right panel) among malignant NK cells and immune cells (indicated at the bottom) in NKTCL. Horizontal lines of identical color connect cells expressing ligands with cells expressing corresponding cognate receptors, and vertical lines highlight the expression patterns of chemokines in selected cell clusters. Cell clusters and chemokines are indicated at the x‐ and y‐axis, respectively. B) Dot plot showing the expression of DPP4 among malignant and normal NK cell clusters (x‐axis). Circle sizes represent the proportions of cells expressing DPP4, and filled colors from light gray to deep purple represent normalized expression levels from low to high. C) Western blotting assay showing the protein expression of DPP4 and ACTIN in the supernatants and cell lysates of NKTCL cell lines (YT and NK‐92) with external DPP4 protein as positive control. D) Multiplex IF staining for DPP4‐expressing malignant NK cells (CD56+DPP4+) in NKTCL biopsies from the SC‐cohort. CD56 and DPP4 proteins as well as nuclear DNA are detected with different colors as indicated on top. Images are representative of biological replicates from three patients. Scale bars, 100 µm. E) Experimental design for the transwell assay to determine the migration ability of NK cells. DPP4 protein and the supernatants from NKTCL cells (YT and NK‐92) were incubated with DPP4 inhibitor (DPP4i) or DMSO as a control for 1 h, which were then incubated with culture medium without chemokines (PBS) or containing chemokine mixture for a 6‐h pretreatment and subsequently added into lower chambers. Peripheral NK cells isolated from PBMCs were then placed into the upper chambers for migration test. Bar plots showing the effect of F) exogenous or G) endogenous DPP4 on the transwell migration rates of NK cells. For (F), NK cells were cultured with chemokine mixture (CXCLs), and/or DPP4, and/or the DPP4i or without these factors (Control) in the lower chambers. For (G), NK cells were cultured with the supernatants from NKTCL cell lines (YT and NK‐92) with or without CXCLs and DPP4i in the lower chambers. Migration rates represent the percentages of migrated cells in all NK cells. Comparisons were made using paired Student's t‐test, and results are shown as mean value ± SD. H) Multiplex IF staining for DPP4 protein in malignant NK cells (CD56+) and extracellular regions as well as related chemokines (CXCL2, CXCL9, and CXCL10) in NKTCL tissue samples. The samples were categorized into two groups based on DPP4 expression: DPP4‐high (left panel) and DPP4‐low (right panel). Images are representative of biological replicates from three patients. Scale bars, 20 µm.
Fig 2: Schematic diagrams of cross‐talks among malignant NK cells and immune cells in the TME of NKTCL. EBV‐infected malignant NK cells and immune cells together participate in the development of NKTCL. 1) Upon EBV infection, LMP1 may contribute to the malignant transformation of NK cells (Figure 2H). 2) Malignant NK cells and TAMs secret a variety of chemokines (including CCL2, CCL3, CCL4, CCL5, etc.) and thereby recruit multiple types of immune cells from peripheral blood through corresponding chemotactic interactions (Figure 3A). 3) Soluble DPP4 secreted by malignant NK cells can truncate and rapidly degrade CXCL2, CXCL9, and CXCL10 in NKTCL TME, whereby hampering the recruitment of CXCR2+CXCR3+ immune cells (Figure 3B–H). 4) Malignant NK cells (especially LMP1+ ones) expressing CD86 and PD‐L1 can negatively regulate the immune response of tumor‐infiltrating T cells including exhausted and regulatory T cells (CD8+ TEX, CD4+ TEX, and Treg; Figures 4A–C and 5A–D). 5) TAMs not only secrete immunosuppressive IL10 and angiogenic VEGFA, but also interact with tumor‐infiltrating T cells through suppressive interactions of CD86‐CTLA4 and PDL1‐PD1 (Figures 4B and 5A).
Fig 3: Experimental investigation of the CXCL2 --> CD8 T effector memory cell_DPP4 interaction found in the IgAN kidney cell-cell communication analysis.a) Flow cytometry analysis of CD8+ memory T cells.b) Identification of CD8+ memory T cells as predominantly effector memory (EM) subtype (CD45RO+ and CD62L−), with a minor fraction of central memory (CM) cells (CD45RO+ and CD62L+).c) Cartoon depiction of the chemotaxis assay using the transwell; overtime, cells inputted to the top chamber of the transwell may be chemoattracted to the bottom chamber, similar to how within tissues, a chemotactic gradient chemoattracts cells to a site of inflammation.d) CXCL2 transwell migration assay conducted on day 0 revealed chemotaxis of CD8+ memory T cells towards CXCL2 (at all dilutions examined for both donors, with the exception of 10 ng/ml for donor 2), consistent with the meta-analysis findings.e-f) In CD8+ memory T cells, reduced DPP4 expression was observed only in the DPP4 KO group, while the wild-type (WT), electroporation only, and non-target control (NTC) KO groups showed no significant alterations on day 11.g) Subsequent CXCL2 transwell migration assay conducted with CD8+ memory T cells to assess the impact of DPP4 KO on CXCL2-induced chemotaxis. DPP4 KO cell’s CXCL2-induced migration was not significantly different from the medium-only condition (for both donors), contrasting with wild-type (WT) and NTC cells in donor 1 and 2 respectively, that showed significantly stronger migration towards CXCL2 compared to the medium-only control (p < 0.05). CXCL12 positive control showed similar levels of significantly higher migration compared to the medium-only control (p < 0.05 for both donors) for all groups (KO, WT, NTC).* p < 0.05. ** p < 0.01, *** p < 0.0001 compared to the medium control condition per donor.
Fig 4: Experimental investigation of the CXCL2 to CD8 T effector memory cell DPP4 interaction found in the IgAN kidney cell-cell communication analysis. (A) Flow cytometry analysis of CD8+ memory T cells. (B) Identification of CD8+ memory T cells as predominantly effector memory (EM) subtype (CD45RO+ and CD62L-), with a minor fraction of central memory (CM) cells (CD45RO+ and CD62L+). (C) Cartoon depiction of the chemotaxis assay using the transwell; overtime, cells inputted to the top chamber of the transwell may be chemoattracted to the bottom chamber, similar to how within tissues, a chemotactic gradient chemoattracts cells to a site of inflammation. (D) CXCL2 transwell migration assay conducted on day 0 revealed chemotaxis of CD8+ memory T cells towards CXCL2 (at all dilutions examined for both donors, except for 10 ng/mL for donor 2), consistent with the meta-analysis findings. (E, F) In CD8+ memory T cells, reduced DPP4 expression was observed only in the DPP4 KO group, while the wild-type (WT), electroporation only, and non-target control (NTC) KO groups showed no significant alterations on day 11. (G) Subsequent CXCL2 transwell migration assay conducted with CD8+ memory T cells to assess the impact of DPP4 KO on CXCL2-induced chemotaxis. DPP4 KO cell’s CXCL2-induced migration was not significantly different from the medium-only condition (for both donors), contrasting with wild-type (WT) and NTC cells in donor 1 and 2 respectively, that showed significantly stronger migration towards CXCL2 compared to the medium-only control (p < 0.05). CXCL12 positive control showed similar levels of significantly higher migration compared to the medium-only control (p < 0.05 for both donors) for all groups (KO, WT, NTC). *p < 0.05. **p < 0.01, ***p < 0.0001 compared to the medium control condition per donor.
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