Fig 1: Mechanisms of IL-33-stimulated macrophages in protection of HPMCs from TIL-executed killing. A) In solid tumors, T cells recognize cancer cells through interaction between specific receptor molecules expressed on the T cells such as TCR and NKG2D, and antigens such as MHC class I and MICA/B. T cells and the targeted cancers form rosette-like flower structures, which permit specific cancer cell killing by T cells. B) In the tumor microenvironment, various stromal cellular components, including cancer-associated fibroblasts (CAFs), tumor-associated macrophages, and cells on the vessel wall coexist and undergo relentlessly changes. These stromal cellular components interact with each other to support tumor growth. For example, perivascular cells and CAFs produce high levels of IL-33 in TME and IL-33 stimulates the conversion of M1 macrophages to become the M2 type through the ST2 receptor expressed in macrophages. The IL-33-activated macrophages produce exceptionally high levels of MMP-9. MMP9 acts as an immunosuppressive sheddase to cleave NKG2D on T cells and MICA/B on cancer cells. Ablation of NKG2D and MICA/B abolishes the formation of rosettes between T cells and cancer cells, thus debilitating the T cell-mediated cancer killing effects.
Fig 2: MMP-9 inhibition restores TIL-mediated cancer killing in vivo. Micrographs of zebrafish with HPMCs (green), IL-33-stimulated macrophages (blue) plus STILs (red) at 0 and 24 h post-implantation. A–H) White arrows point to injected cells. A) Co-injection of STILs, HPMCs, and IL-33-MMCs with SB-3CT (5:1:2). B) Co-injection of STILs, HPMCs, and IL-33-HMCs with SB-3CT (5:1:2). C) Co-injection of STILs, HPMCs, and IL-33-MMCs with siMmp-9 (5:1:2). D) Co-injection of STILs, HPMCs, and IL-33-HMCs with siMMP-9 (5:1:2). E) STILs treated with IL-33-MMCCM with SB-3CT plus HPMCs (5:1). F) STILs treated with IL-33-HMCCM with SB-3CT plus HPMCs (5:1). G) STILs plus HPMCs treated with IL-33-MMCCM with SB-3CT (5:1). H) STILs plus HPMCs treated with IL-33-HMCCM with SB-3CT (5:1). Quantification of calcein-positive areas in the zebrafish and killing rates of TILs were calculated (A, n = 26 samples per group; B, n = 18 samples per group; C, n = 18 samples per group; D, n = 21 samples per group; E, n = 22 samples per group; F, n = 14 samples per group; G, n = 14 samples per group; H, n = 14 samples per group). Data are mean determinants ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, Unpaired Student's t-test.
Fig 3: SP-8356 inhibits matrix-metalloproteinase (MMP) activity at 2-week after alkali burn. (A) Representative image of MMP activity, which is visualized with in situ zymography. Scale bar, 100 µm (magnification, 200×). (B) Representative image of MMP-9 gelatin acrylamide gel zymography. (C) Quantitative analysis of the relative level of MMP-9 activity in whole corneal lysates (n = 9 for sham, n = 12 for saline, n = 9 for HA, n = 9 for SP-8356/HA, n = 10 for PA). MMP-9 activities are shown as means ± SD (*** p < 0.001 vs. saline. # p < 0.05 vs. HA).
Fig 4: MMP-9 mediates the IL-33-macrophage-instigated immunosuppression. A) Heatmap of a myriad of MMPs genes by genome-wide expression profiling of IL-33-MMCs (n = 3 samples per group). B) qPCR quantification of mouse Mmp-9 and human MMP-9 mRNA expression levels of IL-33-stimulated MMCs and HMCs (n = 6 samples per group). Randomized micrographs of calcein-labeled HPMCs (green), DiI-labeled STILs (red), or plus DiD-labeled macrophages (blue) were collected from each sample. Various combinations of cells were co-incubated for 24 h. C–F) Yellow arrows point to TILs, white arrowheads indicate HPMCs, and white arrows point to macrophages. C) Coculturing STILs, HPMCs, and IL-33-MMCs with or without SB-3CT (10:1:2). D) Coculturing STILs, HPMCs, and IL-33-HMCs with or without SB-3CT (10:1:2). E) Coculturing STILs, HPMCs, and IL-33-MMCs with or without siMmp-9 (10:1:2). F) Coculturing STILs, HPMCs, and IL-33-HMCs with or without siMMP-9 (10:1:2). Calcein-positive HPMCs in all groups are quantified (n = 6 random fields per group, 10× magnification). Scale bar = 50 µm. FACS analysis of calcein-positive HPMCs in each group was also represented. Red arrows indicate calcein positive cells. Data are mean determinants ± SEM; n = 3 samples per group. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, Unpaired Student's t-test.
Fig 5: MMP-9 cleaves NKG2D in TILs and MICA/B in HPMCs. Purified STILs were stimulated with rhMMP-9 (5 µg mL-1) in the presence or absence of 20 µm SB-3CT for 24 h. A) NKG2D expression of STILs were analyzed by FACS. B) Fold changes of flow cytometry mean fluorescence intensity (MFI) values were quantified. HPMCs were stimulated with rhMMP-9 in the presence or absence of 20 µm SB-3CT for 24 h. C) MICA/B expression levels in HPMCs were analyzed by FACS. D) Fold changes of flow cytometry MFI values were quantified. All experiments were repeated five times. Data are mean determinants ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 versus controls; NS, not significant, Unpaired Student's t-test.
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