Low miR-16 expression induces regulatory CD4+NKG2D+ T cells involved in colorectal cancer progression

Abstract

MiR-15a/16 is a member of the miRNA cluster that exhibits tumor suppression and immune modulation via targeting multiple genes. Decreased miR-15a/16 expression is involved in many cancer cells. Here, miR-16 had decreased expression in NK1.1^-CD4⁺NKG2D⁺ T cells and bound with the 3'-UTR of NKG2D gene. MiR-15a/16-deficient mice had many CD4⁺NKG2D⁺ T cells, which produced TGF-β1 and IL-10 and inhibited the IFN-γ production of CD8⁺ T cells. Adoptive transfer of NK1.1^-CD4⁺NKG2D⁺ T cells from miR-15a/16-deficient mice promoted tumor growth in vivo. However, no changes for NK1.1^-CD4⁺NKG2D⁺ T cells were found in the miR-15a/16-transgenic mice. Although the miR-15a/16 transgenic mice transplanted with B16BL6 or MC38 cells exhibited rapid growth, these tumor-bearing mice did not show changes in NK1.1^-CD4⁺NKG2D⁺ T cell distributions in either spleens or tumors. When NK1.1^-CD4⁺ T cells were stimulated by α-CD3/sRAE-1 ex vivo, the NKG2D expression was difficult to induce in the T cells of miR-15a/16-transgenic mice. Finally, increased frequencies of regulatory CD4⁺NKG2D⁺ T cells with low miR-16 levels were observed in patients with late-stage colorectal cancer (Duke's C, D). Thus, miR-16 modulates NK1.1^-CD4⁺NKG2D⁺ T cell functions via targeting NKG2D. Low miR-16 expression in CD4⁺ T cells induces the regulatory CD4⁺NKG2D⁺ T subpopulation, which promotes tumor evasion via the secretion of immune-suppressive molecules.

Keywords: CD4+ T cell; NKG2D; colorectal cancer; miR-15a/16; regulation.

Figure 1

Decreased miR-16 expression in CD4⁺NKG2D⁺ T cells induced by α-CD3/sRAE-1. A, B. NKG2D expression of NK1.1^-CD3⁺CD4⁺ cells induced by different treatments. C. MiR-16 levels in CD4⁺NKG2D⁺ T cells induced by various stimulations as detected by real-time PCR. D. NKG2D expression on NK1.1^-CD3⁺CD4⁺ cells induced by various cytokines. E. NKG2D transcriptions on NK1.1^-CD3⁺CD4⁺ cells treated by cytokines. F. Variations of miR-16 levels in CD4⁺NKG2D⁺ T cells. All experiments were repeated twice. *, P < 0.05; **, P < 0.01; ns, no significance.

Figure 2

MiR-16 binding with NKG2D 3’-UTR. A. Nucleotide pairs of miR-16 and NKG2D 3’-UTR. B. Cloning of native and mutated 3’-UTR into the pGL3 vector. C. Luciferase reporter assay with miR-16 mimics and recombinant pGL3 plasmids co-transfection. Transfection experiments were conducted three times. *, P < 0.05; **, P < 0.01.

Figure 3

Increased regulatory CD4⁺NKG2D⁺ T cells in miR-15a/16^-/- mice. A. Frequencies of CD3⁺NK1.1^-CD4⁺NKG2D⁺ T cells in the spleens of miR-15a/16^-/- mice. B. NKG2D expression on NK1.1^-CD3⁺CD4⁺ cells. C. TGF-β1 and IL-10 production of CD4⁺NKG2D⁺ T cells. D. IFN-γ secretion of CD8⁺ T cells after coculture with CD4⁺NKG2D⁺ T cells with or without α-TGF-β1 (10 μg/ml). E. Growth curve of MC38 tumors in mice transfused with CD4⁺NKG2D⁺ T cells. *, P < 0.05; **, P < 0.01.

Figure 4

Variations of CD4⁺NKG2D⁺ T cells in miR-15a/16-transgenic mice. (A) Diagram of the gene element for microinjection. (B) MiR-16 levels in the tissues of gut, lung, liver, and stomach. (C) GFP⁺ cells in tail veins of transgenic mice. (D) MiR-16 levels of CD4⁺NKG2D⁺ T cells in WT, KO, and TG mice. (E) Transcriptional levels of NKG2D in CD4⁺ T cells of three mouse groups. (F) NKG2D protein levels on CD4⁺ T cells of three groups of mice. (G) Frequencies of CD4⁺CD69⁺, CD4⁺CD44⁺, CD4⁺CD25⁺, and CD4⁺CD28⁺ T cells in three mouse groups. Growth of transplanted B16BL6 (H) and MC38 (I) cells in the transgenic mice and distributions of CD4⁺NKG2D⁺ T cells in spleens and tumor tissues. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.

Figure 5

Inefficient induction of miR-15a/16^TG-CD4⁺NKG2D⁺ T cells ex vivo. A. Induced CD4⁺NKG2D⁺ T cells of TG mice by α-CD3/sRAE-1. B. TGF-β1 expression on CD4⁺ T cells treated by α-CD3/sRAE-1. C. Inductions of CD4⁺CD69⁺, CD4⁺CD44⁺, CD4⁺CD28⁺, CD4⁺CD25⁺, and CD4⁺IL-10⁺ cells. All experiments were conducted at least three times. *, P < 0.05; **, P < 0.01.

Figure 6

CD4⁺NKG2D⁺ T cells with low miR-16 level involved in CRC progression. (A) NKG2D expression of CD4⁺ T cells in patients with CRC detected by flow cytometry. (B) Elevated NKG2D expression of CD4⁺ T cells indicated by statistical analysis. (C) CD4⁺NKG2D⁺ T cell frequencies in patients with CRC of Duke’s A-D stages. (D) Correlation of miR-16 level with NKG2D MFI in CD4⁺NKG2D⁺ T cells. (E) Comparison of miR-16 levels between CD4⁺NKG2D⁺ and CD4⁺NKG2D^- T cells in the same individual. IL-10 (F) and TGF-β1 (G) production of CD4⁺NKG2D⁺ T cells in different CRC stages. (H) Correlation of miR-16 level with IL-10 MFI (Left) or TGF-β1 MFI (Right) in CD4⁺NKG2D⁺ T cells of patients with CRC. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.