Interleukin-33 is a Novel Immunosuppressor that Protects Cancer Cells from TIL Killing by a Macrophage-Mediated Shedding Mechanism

Abstract

Recognition of specific antigens expressed in cancer cells is the initial process of cytolytic T cell-mediated cancer killing. However, this process can be affected by other non-cancerous cellular components in the tumor microenvironment. Here, it is shown that interleukin-33 (IL-33)-activated macrophages protect melanoma cells from tumor-infiltrating lymphocyte-mediated killing. Mechanistically, IL-33 markedly upregulates metalloprotease 9 (MMP-9) expression in macrophages, which acts as a sheddase to trim NKG2D, an activating receptor expressed on the surface of natural killer (NK) cells, CD8+ T cells, subsets of CD4+ T cells, iNKT cells, and γδ T cells. Further, MMP-9 also cleaves the MHC class I molecule, cell surface antigen-presenting complex molecules, expressed in melanoma cells. Consequently, IL-33-induced macrophage MMP-9 robustly mitigates the tumor killing-effect by T cells. Genetic and pharmacological loss-of-function of MMP-9 sheddase restore T cell-mediated cancer killing. Together, these data provide compelling in vitro and in vivo evidence showing novel mechanisms underlying the IL-33-macrophage-MMP-9 axis-mediated immune tolerance against cancer cells. Targeting each of these signaling components, including IL-33 and MMP-9 provides a new therapeutic paradigm for improving anticancer efficacy by immune therapy.

Keywords: T-cell receptors; cancer cells; cytolytic T cells; interleukin-33; metalloprotease.

Figure 1

Debilitation of TIL‐mediated tumor killing by IL‐33‐primed macrophages. A–F) Randomized micrographs of calcein‐labeled HPMCs (green), DiI‐labeled NSTILs (red) or STILs (red), or plus DiD‐labeled macrophages (blue) were collected from each sample. Various combinations of cells were co‐incubated for 24 h. Yellow arrows point to TILs, white arrowheads indicate HPMCs, and white arrows point to macrophages. A) NTILs plus HPMCs (10:1). B) STILs plus HPMCs (10:1). C) Coculturing STILs, HPMCs, and MMCs (10:1:2). D) Coculturing STILs, HPMCs, and IL‐33‐stimulated MMCs (10:1:2). E) Coculturing STILs, HPMCs, and HMCs (10:1:2). F) Coculturing STILs, HPMCs, and IL‐33‐stimulated HMCs (10:1:2). Calcein‐positive HPMCs in all groups are quantified (n = 6 random fields per group, 10× magnification). Scale bar = 50 µm. G) FACS analysis of calcein‐positive HPMCs in samples containing NSTILs and STILs. H) FACS analysis of calcein‐positive HPMCs in samples containing STILs plus MMCs or IL‐33‐stimulated MMCs. I) FACS analysis of calcein‐positive HPMCs in samples containing STILs plus HMCs or IL‐33‐stimulated HMCs. Arrows in (G)–(I) 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.

Figure 2

Soluble fraction of IL‐33‐stimulated macrophage mediates immunosuppression. A–D) Randomized micrographs of calcein‐labeled HPMCs (green) and DiI‐labeled STILs (red) that were non‐treated or treated with conditioned media derived from the IL‐33‐stimulated macrophages. Various combinations of cells were co‐incubated for 24 h. Yellow arrows point to STILs and white arrowheads indicate HPMCs. A) STILs were treated with IL‐33‐MMCCM for 24 h plus HPMCs (10:1). B) STILs were treated with IL‐33‐HMCCM for 24 h plus HPMCs (10:1). C) Coculturing STILs and HPMCs were treated with IL‐33‐MMCCM for 24 h (10:1). D) Coculturing STILs and HPMCs treated with IL‐33‐HMCCM for 24 h (10:1). Calcein‐positive HPMCs in all groups are quantified (n = 6 random fields per group, 10× magnification). Scale bar = 50 µm. Data are mean determinants ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, Unpaired Student's t‐test.

Figure 3

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.

Figure 4

IL‐33‐stimulated macrophages protect HPMCs from TIL killing in zebrafish. A–F) Representative micrographs of calcein‐labeled HPMCs (green), DiI‐labeled TILs (red), or plus DiD‐labeled macrophages (blue) were collected at 0 and 24 h after co‐implantation into the zebrafish. White arrows point to injected cells. A) NTILs plus HPMCs (5:1). B) STILs plus HPMCs (5:1). C) Co‐injection of STILs, HPMCs, and MMCs (5:1:2). D) Co‐injection of STILs, HPMCs, and IL‐33‐stimulated MMCs (5:1:2). E) Co‐injection of STILs, HPMCs, and HMCs (5:1:2). F) Co‐injection of STILs, HPMCs, and IL‐33‐stimulated HMCs (5:1:2). Dashed lines rectangular and amplify the indicated regions. (Scale bars = 200 µm; amplified fields, 100 µm). Quantification of calcein‐positive areas in the zebrafish and killing rates of TILs were calculated (A, n = 15 samples per group; B, n = 14 samples per group; C, n = 23 samples per group; D, n = 25 samples per group; E, n = 20 samples per group; F, n = 19 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.

Figure 5

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.

Figure 6

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.

Figure 7

IL‐33‐stimulated macrophages ablate rosette formation between TILs and HPMCs. Randomized micrographs of calcein‐labeled HPMCs (green), DiI‐labeled NSTILs (red) or STILs (red), or plus DiD‐labeled macrophages (blue) were collected from each sample. Various combinations of cells were co‐incubated for 24 h. A) NTILs plus HPMCs (10:1). B) STILs plus HPMCs (10:1). C) Coculturing STILs, HPMCs, and HMCs (10:1:2). D) Coculturing STILs, HPMCs, and IL‐33‐stimulated HMCs (10:1:2). E) STILs treated with IL‐33‐HMCCM for 24 h plus HPMCs (10:1). F) STILs treated with IL‐33‐HMCCM containing 20 µM SB‐3CT plus HPMCs (10:1). G) Coculturing STILs and HPMCs treated with IL‐33‐HMCCM for 24 h (10:1). H) STILs plus HPMC treated with IL‐33‐HMCCM containing 20 µM SB‐3CT (10:1). I) Coculturing STILs with siRNA‐control and HPMCs for 24 h (10:1). J) Coculturing STILs with siRNA‐NKG2D and HPMCs for 24 h (10:1). K) Coculturing HPMCs with siRNA‐control and STILs for 24 h (10:1). L) Coculturing HPMCs with siRNA‐MICA/B and STILs for 24 h (10:1). M) The rosette formations were assessed and calculated (n = 6 random fields per group, 10× magnification). Scale bar = 50 µm. Dashed lines rectangular and amplify the indicated regions. Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, Unpaired Student's t‐test.

Figure 8

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.