Accelerated tumor growth in mice deficient in DNAM-1 receptor

Since the identification of ligands for human and mouse DNAM-1, emerging evidence has suggested that DNAM-1 plays an important role in the T cell– and natural killer (NK) cell–mediated recognition and lysis of tumor cells. However, it remains undetermined whether DNAM-1 is involved in tumor immune surveillance in vivo. We addressed this question by using DNAM-1–deficient mice. DNAM-1–deficient cytotoxic T lymphocyte (CTL) and NK cells showed significantly less cytotoxic activity against DNAM-1 ligand-expressing tumors in vitro than wild-type (WT) cells. The methylcholanthrene (MCA)-induced fibrosarcoma cell line Meth A expressed the DNAM-1 ligand CD155, and DNAM-1–deficient mice showed increased tumor development and mortality after transplantation of Meth A cells. Moreover, the DNAM-1–deficient mice developed significantly more DNAM-1 ligand-expressing fibrosarcoma and papilloma cells in response to the chemical carcinogens MCA and 7,12-dimethylbenz[a]anthracene (DMBA), respectively, than did WT mice. These results indicate that DNAM-1 plays an important role in immune surveillance of tumor development

Increased Soluble CD155 in the Serum of Cancer Patients

Emerging evidence suggests that DNAM-1 (CD226) play an important role in the recognition of tumor cells and their lysis by cytotoxic T lymphocytes (CTL) and NK cells. Although the DNAM-1 ligand CD155 is ubiquitously expressed in various tissues, many human tumors significantly upregulate the expression of CD155; DNAM-1 on CTL and NK cells may be involved in tumor immunity. However, unlike those in mice, human tissues also express soluble isoforms of CD155 (sCD155) that lack the transmembrane region. Here, we show that sCD155 levels were significantly higher in the sera of 262 patients with lung, gastrointestinal, breast, and gynecologic cancers than in sera from healthy donors. In addition, the sCD155 levels were significantly higher in patients with early stage (stages 1 and 2) gastric cancer than in healthy donors, and were significantly higher in patients withadvanced stage (stages 3 and 4) disease than in patients in those with early stage disease and healthy donors. Moreover, the sCD155 levels were significantly decreased after surgical resection of cancers. Thus, sCD155 level in serum may be potentially useful as a biomarkerfor cancer development and progression

Increased CD112 Expression in Methylcholanthrene-Induced Tumors in CD155-Deficient Mice

<div><p>Tumor recognition by immune effector cells is mediated by antigen receptors and a variety of adhesion and costimulatory molecules. The evidence accumulated since the identification of CD155 and CD112 as ligands for DNAM-1 in humans and mice has suggested that the interactions between DNAM-1 and its ligands play an important role in T cell– and natural killer (NK) cell–mediated recognition and lysis of tumor cells. We have previously demonstrated that methylcholanthrane (MCA) accelerates tumor development in DNAM-1–deficient mice, and the <i>Cd155</i> level on MCA-induced tumors is significantly higher in DNAM-1–deficient mice than in wild-type (WT) mice. By contrast, <i>Cd112</i> expression on the tumors is similar in WT and DNAM-1-deficient mice, suggesting that CD155 plays a major role as a DNAM-1 ligand in activation of T cells and NK cells for tumor immune surveillance. To address this hypothesis, we examined MCA-induced tumor development in CD155-deficient mice. Unexpectedly, we observed no significant difference in tumor development between WT and CD155-deficient mice. Instead, we found that <i>Cd112</i> expression was significantly higher in the MCA-induced tumors of CD155-deficient mice than in those of WT mice. We also observed higher expression of DNAM-1 and lower expression of an inhibitory receptor, TIGIT, on CD8⁺ T cells in CD155-deficient mice. These results suggest that modulation of the expression of receptors and CD112 compensates for CD155 deficiency in immune surveillance against MCA-induced tumors.</p></div

Relative cytokine mRNA levels in MCA-induced tumors.

<p>(A, B) Fibrosarcomas induced by 5 µg MCA in WT or CD155-deficient (KO) C57BL/6N (A) or BALB/c (B) mice were resected from each mouse and subjected to quantitative qRT-PCR for transcripts of the indicated cytokines. Horizontal bars represent means and error bars represent means ± SEM. <i>P</i> Values for Student’s <i>t</i> test are shown.</p

A hypothetical model for interactions between activating/inhibitory receptors DNAM-1, TIGIT and CD96 on T or NK cells and CD155/CD112 ligands expressed on MCA-induced fibrosarcoma in WT or CD155-deficient mice.

<p>(A) DNAM-1 and TIGIT bind to both of CD155 and CD112, while CD96 interacts with CD155 only. Each receptor-ligand interaction transduces either activating or inhibitory signal, as shown by the red or blue arrow, respectively. The modulation of the receptors and ligand expression on CD155-deficient (KO) fibrosarcoma are indicated. (B) The sums of the activating and inhibitory signals are similar between WT and KO.</p

MCA-induced tumor development in WT and CD155-deficient mice.

<p>(A, B) WT (<i>n</i> = 18 and 16, respectively) or CD155-deficient (KO; <i>n</i> = 19 and 20, respectively) C57BL/6N mice were injected s.c. with 5 µg (A) or 100 µg (B) methylcholanthrane (MCA) on day 0. (C) WT (<i>n</i> = 15) or KO (<i>n</i> = 10) BALB/c mice were injected s.c. with 5 µg MCA on day 0. Tumor size in each mouse was measured once a week. Tumor size (top) and survival data (bottom) are shown.</p

Expression of CD155 ligands on resting and activated T cells from WT and CD155-deficient mice.

<p>(A) Peripheral blood lymphocytes from WT (<i>n</i> = 3) or CD155-deficient (KO; <i>n</i> = 3) C57BL/6N mice were stained with anti-DNAM-1, anti-TIGIT, or anti-CD96 antibodies (WT; blue lines, KO; red lines) or control antibodies (WT; light blue lines, KO; pink lines), and analyzed by flow cytometry. The numbers (WT; in blue, KO; in red) indicate the mean fluorescence intensity (MFI) of DNAM-1, TIGIT, and CD96 staining. Representative data are shown. (B) MFI was used to analyze DNAM-1 expression on CD4⁺ T, CD8⁺ T, and NK cells as in (A). Error bars represent means ± SD. (C) CD8⁺ T cells purified from spleen were activated with anti-CD3 antibody and IL-2 for the indicated number of days. Cells were stained and analyzed by flow cytometry, as described in (A). (D) The expression of DNAM-1, TIGIT, and CD96 on CD8⁺ T cells is shown as MFI as in (C). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Relative <i>Cd112</i> mRNA levels in organs and MCA-induced tumors.

<p>(A) Tissues from WT (<i>n</i> = 3) or CD155-deficient (KO; <i>n</i> = 3) mice were subjected to quantitative qRT-PCR for <i>Cd112</i>. Error bars represent means ± SD. (B, C) Fibrosarcomas induced in WT or CD155-deficient C57BL/6N (B) or BALB/c (C) mice by 5 µg MCA were resected from each mouse and subjected to qRT-PCR for <i>Cd112</i>. Horizontal bars represent means and error bars represent means ± SEM. <i>P</i> Values for Student’s <i>t</i> test are shown.</p