Intraepithelial Lymphocytes Suppress Intestinal Tumor Growth by Cell-to-Cell Contact via CD103/E-Cadherin Signal

Abstract

Background & aims: The reason why small intestinal cancer is rarer than colorectal cancer is not clear. We hypothesized that intraepithelial lymphocytes (IELs), which are enriched in the small intestine, are the closest immune cells to epithelial cells, exclude tumor cells via cell-to-cell contact.

Methods: We developed DPE-green fluorescent protein (DPE-GFP) × adenomatous polyposis coli; multiple intestinal neoplasia (APC^min ) mice, which is a T-cell-reporter mouse with spontaneous intestinal tumors. We visualized the dynamics of IELs in the intestinal tumor microenvironment and the interaction between IELs and epithelial cells, and the roles of cell-to-cell contact in anti-intestinal tumor immunity using a novel in vivo live-imaging system and a novel in vitro co-culture system.

Results: In the small intestinal tumor microenvironment, T-cell movement was restricted around blood vessels and the frequency of interaction between IELs and epithelial cells was reduced. Genetic deletion of CD103 decreased the frequency of interaction between IELs and epithelial cells, and increased the number of small intestinal tumors. In the co-culture system, wild-type IELs expanded and infiltrated to intestinal tumor organoids from APC^min mice and reduced the viability of them, which was cell-to-cell contact and CD103 dependent.

Conclusions: The abundance of IELs in the small intestine may contribute to a low number of tumors, although this system may not work in the colon because of the sparseness of IELs. Strategies to increase the number of IELs in the colon or enhance cell-to-cell contact between IELs and epithelial cells may be effective for the prevention of intestinal tumors in patients with a high cancer risk.

Keywords: IELs; In Vivo Live Imaging; Organoid.

Graphical abstract

Figure 1

Visualization of 3D distribution of T cells in the microenvironment of small intestinal tumors shows T cells localized around blood vessels in the tumor. (A) Experimental design. Wide area images of transparent small intestinal tissues of (B) DPE–GFP × APC^min or (C) DPE–GFP mice. Green, GFP; blue, Hoechst 33342; red, dextran-tetramethylrhodamine. Tomographic images of the x-axis and y-axis of small intestinal tissues of (D) DPE–GFP × APC^min or (E) DPE–GFP mice. Tomographic images of the x-axis and z-axis of small intestinal tissues of (F) DPE–GFP × APC^min or (G) DPE–GFP mice. 3D images of transparent small intestine of (H) DPE–GFP × APC^min or (I) DPE–GFP mice. (J) Number of T cells and (K) percentage of T cells in contact with blood vessel in the region of interest (ROI) (n = 3, each). Graphs show means ± SEM. ∗P < .05. MPE, multi-photon excitation microscopy.

Figure 2

Visualization of T-cell dynamics in the intestinal tumor microenvironment by an in vivo live-imaging system with the 2-photon microscope. (A) Experimental design. Wide area images of live small intestinal tissues of (B) DPE–GFP × APC^min or (C) DPE–GFP mice. Green, GFP; blue, Hoechst 33342; red, intravenous dextran-tetramethylrhodamine; yellow, intraluminal dextran–Alexa Fluor 647. Histologic findings of small intestinal tissues of (D) DPE–GFP × APC^min or (E) DPE–GFP mice after in vivo live imaging. Z-stack images of live small intestinal tissues of (F) DPE–GFP × APC^min or (G) DPE–GFP mice. Time-lapse images of IELs of nontumor (N-T) region in (H) DPE–GFP × APC^min or (I) DPE–GFP mice. (J) Speed, (K) track length, (L) confinement ratio, and (M) total time of GFP⁺ cells in contact with IECs were calculated (n = 3, each). Graphs show means ± SEM. Data were pooled from 3 independent experiments. SI, small intestinal.

Figure 3

Cell-to-cell contact between small intestinal-IELs and epithelial cells is decreased in the intestinal tumor microenvironment. (A) Representative image of live small intestinal tissue with both tumor region and nontumor region in DPE–GFP × APC^min mice. Time-lapse images of (B) tumor region and (C) nontumor (N-T) region of DPE–GFP × APC^min mice. Green, GFP; blue, Hoechst 33342; red, intravenous dextran-tetramethylrhodamine; yellow, intraluminal dextran-Alexa Fluor 647. (D) Number, (E) speed, (F) track length, (G) confinement ratio, and (H) total time of GFP⁺ cells in contact with IECs (n = 8, each). (I) Percentage of GFP⁺ cells in contact with blood vessels (n = 10). Graphs show means ± SEM. Data were pooled from 8 independent experiments for panels D–H and are representative of similar independent 3 experiments for panel I. Histologic immunohistochemistry findings of small intestinal tissue of (J) DPE–GFP × APC^min or (K) DPE–GFP mice. Red, E-cadherin; blue, 4’,6-diamidino-2-phenylindole (DAPI). ∗P < .05.

Figure 4

Visualization of the T-cell dynamics in small intestinal tumors. Still images of the 3 representative cases of in vivo live-imaging of the small intestinal tumors and T cells in the DEP–GFP × APC^min mice.

Figure 5

Expression of CD103 in T cells in the small intestine. (A) Representative histograms for CD103 gating in each group. Percentages of CD103⁺ cells in CD3⁺ and CD3^- cells in (B) small intestinal (SI)-IELs or (C) SI-LPLs. Cells were recovered from tumor and nontumor (N-T) regions of APC^min mice and control WT mice (n = 3, each). Graphs show means ± SEM.

Figure 6

Cell-to-cell contact between small intestinal (SI)-IELs and epithelial cells is decreased in Itgae^-/-mice. Number of T cells (CD3⁺ cells), TCR γδ (CD3⁺TCRγδ⁺TCRβ^- cells), DN (CD3⁺TCRβ⁺CD4^-CD8β^- cells), CD4⁺ (CD3⁺TCRβ⁺CD4⁺CD8β^- cells), and CD8⁺ (CD3⁺TCRβ⁺CD4^-CD8β⁺ cells) in (A) SI-IELs and (B) SI-LPLs, and (C) regulatory T cells (Tregs) (CD3⁺CD4⁺CD25⁺Foxp3⁺ cells) in SI-LPLs of Itgae^-/- or littermate control mice (n = 8 in Itgae^-/- mice and n = 9 in control mice). Graphs show means ± SEM. Data were pooled from 7 independent experiments. Representative images of live small intestinal tissues of (D) Itgae^-/- × DPE–GFP or (F) control DPE–GFP mice. Time-lapse images of IELs of (E) Itgae^-/- × DPE–GFP or (G) control DPE–GFP mice. Green, GFP; blue, Hoechst 33342; red, intraluminal dextran–Alexa Fluor 594. (H) Speed, (I) track length, (J) confinement ratio, and (K) total time of GFP⁺ cells in contact with IECs (n = 6 each). Graphs show means ± SEM. Data were pooled from 6 independent experiments. ∗P < .05.

Figure 7

The number of intestinal tumors increased in Itgae^-/-mice. (A) Representative images of gross appearance of ileum of Itgae^-/- × APC^min and control APC^min mice. (B) Histologic findings of intestinal tumors of Itgae^-/- × APC^min and control APC^min mice aged 16 weeks. (C) Mean area, (D) total number, and (E) total area of small intestinal tumors in female Itgae^-/- × APC^min and control APC^min mice (n = 10 in Itgae^-/- × APC^min mice and n = 8 in control APC^min mice). Graphs show means ± SEM. Data were pooled from 6 independent experiments. ∗P < .05.

Figure 8

Antitumor immune response was provoked between co-cultured WT-IELs and intestinal tumor organoids. (A) Experimental design. Organoids developed from the small intestine of WT or small intestinal tumor of APC^min mice were co-cultured with IELs from EGFP mice. After 7–9 days of co-culture, they were collected and dispersed into single cells. Numbers of cells were counted using flow cytometry. (B) Representative images of organoids and IELs after 7 days of co-culture. (C) Time-lapse images of APC^min organoids and IELs after 4 days of co-culture. (D) Number of live epithelial cells (7-AAD^-EpCAM⁺CD45^- cells) collected from organoids in each group (n = 6–8). (E) Number of IELs (7-AAD^-GFP⁺ cells) in each group (n = 7–8). Graphs show means ± SEM. Data are representative of 2 similar independent experiments. (F and G) Evaluation of cytokines in the supernatant after 7 days of co-culture of organoids with IELs from WT mice. Concentration of (F) interferon (IFN)-γ and (G) tumor necrosis factor (TNF)-α (n = 6 each). Graphs show means ± SEM. Data were pooled from 2 independent experiments. ∗P < .05. Org, organoid; SI, small intestinal.

Figure 9

Time-lapse images of IELs and organoids. (A and B) Time-lapse images of in vitro co-culture of GFP IELs and (A) APC^min organoids or (B) WT organoids.

Figure 10

Evaluation of proliferation and early apoptosis of epithelial cells recovered from WT or APC^minorganoids co-cultured with or without WT IELs. (A) Percentages of Ki67⁺ cells in all epithelial cells (EpCAM⁺ CD45^- cells) recovered from cultured organoids in each group (n = 6–8). (B) Percentages of early apoptotic cells (AnnexinV⁺7-AAD^- cells) in all epithelial cells recovered from cultured organoids in each group (n = 6–7). Graphs show means ± SEM. Data were pooled from 2 independent experiments. ∗P < .05. Org, organoid.

Figure 11

Evaluation of produced cytokines in the supernatant of in vitro culture of WT or APC^minorganoids with or without WT IELs. Concentration of (A) IL2, (B) IL4, (C) IL17A, (D) IL6, (E) IL10, and (F) TGF-β1 in the supernatant of each group (n = 6 each). Graphs show means ± SEM. Data were pooled from 2 independent experiments. ∗P < .05. Org, organoid.

Figure 12

Immunohistochemistry of E-cadherin in the small intestinal organoids of APC^minor WT mice. Representative images of immunohistochemistry of fixed small intestinal organoids of APC^min or WT mice. Red, E-cadherin; blue, 4’,6-diamidino-2-phenylindole (DAPI).

Figure 13

Co-culture of γδ, DN, CD4⁺, or CD8⁺IELs from WT mice and APC^minorganoids. (A) The number of recovered IELs (7-AAD^-EpCAM^-CD45⁺ cells) in each group. (B) The number of live epithelial cells (7-AAD^-EpCAM⁺CD45^- cells) recovered from APC^min organoids in each group. Graphs show means ± SEM. Data were pooled from 2 independent experiments. n = 7–9 in each group. ∗P < .05. Org, organoid.

Figure 14

Antitumor immune response of IELs against intestinal tumor organoids is cell-to-cell contact-dependent. (A) Experimental design. Organoids developed from a small intestinal tumor of APC^min mice were co-cultured with IELs from EGFP mice with or without a separation insert. (B) Representative images of organoids and IELs after 7 days of co-culture. (C) Number of live epithelial cells (7-AAD^-EpCAM⁺CD45^- cells) collected from organoids in each group (n = 7–8). Graphs show means ± SEM. Data were pooled from 2 independent experiments. (D) Experimental design. Organoids developed from a small intestinal tumor of APC^min mice were co-cultured with IELs from WT or Itgae^-/- mice. (E) Representative images of organoids and IELs after 7 days of co-culture. (F) Number of live epithelial cells (7-AAD^-EpCAM⁺CD45^- cells) collected from organoids in each group (n = 9–11). (G) Number of IELs (7-AAD^-EpCAM^-CD45⁺ cells) in each group (n = 9–11). Graphs show means ± SEM. Data were pooled from 2 independent experiments. ∗P < .05. Org, organoid; SI, small intestinal; T-W, transwell.

Figure 15

Evaluation of cell death of epithelial cells recovered from APC^minorganoids co-cultured with or without IELs. (A) Percentages of 7-AAD⁺ cells in epithelial cells (EpCAM⁺CD45^- cells) recovered from APC^min organoids in each group. Graphs show means ± SEM. Data were pooled from 2 independent experiments. n = 7–8 in each group. (B) Percentages of 7-AAD⁺ cells in epithelial cells (EpCAM⁺CD45^- cells) recovered from APC^min organoids in each group. Graphs show means ± SEM. Data were pooled from 2 independent experiments. n = 9–11 in each group. ∗P < .05. Org, organoid; T-W, transwell.