Cd226-/- natural killer cells fail to establish stable contacts with cancer cells and show impaired control of tumor metastasis in vivo

Abstract

CD226 is an activating receptor expressed on natural killer (NK) cells, CD8⁺ T cells, and other immune cells. Upon binding to its ligands expressed on target cells, CD226 activates intracellular signaling that triggers cytokine production and degranulation in NK cells. However, the role of CD226 in contact dynamics between NK and cancer cells has remained unclear. Our time-lapse images showed that individual wild-type CD226⁺ NK cells contacted B16F10 melanoma cells for 23.7 min, but Cd226^-/- NK cells only for 12.8 min, although both NK cell subsets showed equal contact frequency over 4 h. On the surface of B16F10 cells, CD226⁺ cells stayed at the same site with oscillating movement (named stable contact), while Cd226^-/- NK cells moved around at a velocity of 4 μm/min (named unstable contact). Consequently, Cd226^-/- NK cells did not kill B16F10 cells in vitro and did not inhibit their metastasis into the lung in vivo. Taken together, our data demonstrate that CD226 enables prolonged stable interaction between NK and cancer cells, which is needed for efficient killing of cancer cells.

Keywords: Contact duration; NK cell-mediated cytotoxicity; contact dynamics; contact stability; melanoma.

Figure 1.

Cd226^−/− NK cells show impaired cytotoxicity. (A) Phenotypes of wild-type (WT) NK cells (n = 3). (B) Phenotypes of Cd226^−/− NK cells (n = 3). (C) Phenotypes of B16F10 cells (n = 3). (D) Cytotoxicity of NK cells against B16F10 cells was determined by the LDH assay (n = 3, *p < 0.01). (E) To analyze exocytosis, NK and B16F10 cells were co-cultured for 2 h in the presence of anti-CD107a-FITC antibody, and CD107⁺ expression level on NK cells was determined using flow cytometry (n = 3, *p < 0.01). (F) Intracellular perforin and granzyme B protein levels of wild-type and Cd226^−/− NK cells (n = 3).

Figure 2.

CD226-negative NK cells show impaired cytotoxicity. (A) CD226⁺ and CD226⁻ NK cells were purified from total splenic NK cells using flow cytometry and their cytotoxicity against B16F10 cells was determined by the LDH assay (n = 3, *p < 0.01). (B) To analyze exocytosis, NK and B16F10 cells were co-cultured for 2 h in the presence of anti-CD107a-FITC antibody. CD107⁺ expression level on CD226⁺ and CD226⁻ NK cells was determined using flow cytometry (n = 3). (C) NK cells were treated with isotype, anti-CD226, or anti-NKG2D neutralizing antibodies (10 μg/ml) for 2 h. Cytotoxicity of NK cells against B16F10 cells was determined by the LDH assay (n = 3, *p < 0.01). (D) B16F10 cells were treated with negative control or CD155 siRNAs. CD155 expression level was determined by using flow cytometry and cytotoxicity of NK cells against B16F10 cells was determined by the LDH assay (n = 3, *p < 0.01).

Figure 3.

CD226-deficient NK cells show impaired cytotoxicity at the single cell level. (A) Unstained NK cells (unsorted WT, CD226⁺ WT, CD226⁻ WT, or Cd226^−/− NK cells) and calcein AM–stained B16F10 cells (transfected with CD155 siRNA or not) were imaged every 2 min from 1 h to 5 h. Representative photos are shown (magnification, 200 ×; scale bars, 50 μm, n = 10 movies of 3 independent experiments per group). (B) Representative images show dying of cancer cells (original magnification, 200 ×; electronically zoomed). Cancer cells underwent rounding and propidium (PtdIns) uptake phases. Ratios of rounding and PtdIns uptake cells were counted every 1 h. (C) Time needed for cells to become rounded morphology was calculated (n = 129, 59, 4, 13, and 22 from the left). (D) Time needed for rounded cells to uptake PtdIns was calculated (n = 101, 81, 4, 6, and 21 from the left).

Figure 4.

Migration modes of NK cells around cancer cells. (A) Representative profile of NK cell migration speed (n = 10 movies of 3 independent experiments per group). (B – E) Mean speed (B), track length (C), track straightness (D), and mean square displacement (E) of WT (n = 255) and Cd226^−/− NK cells (n = 174).

Figure 5.

Classification of NK cells based on contact modes. (A) Unstained NK cells (unsorted WT, CD226⁺ WT, CD226⁻ WT, or Cd226^−/− NK cells) and calcein AM–stained B16F10 cells (transfected with CD155 siRNA or not) were imaged every 2 min from 1 h to 5 h. Representative images of the contact modes of CD226⁺ NK cells (arrows) showing free migration, killing contact, and non-killing contact (magnification, 200 ×; electronically zoomed; scale bars, 15 μm; n = 10 movies of 3 independent experiments per group). (B) Ratios of contacting NK cells to total NK cells (n = 1,413, 527, 521, 376, and 688 from the left). (C) Ratios of killing per total contacts (n = 279, 93, 118, 84, and 146 from the left). (D, E) Contact duration of killing and non-killing interactions between cancer cells and unsorted (n = 79), Cd226^−/− (n = 191), CD226⁺ (n = 71), and CD226⁻ (n = 145) NK cells. (n = 3, *p < 0.01).

Figure 6.

Stability of contacts between NK and cancer cells. (A) Unstained NK cells (unsorted WT, CD226⁺ WT, CD226⁻ WT, or Cd226^−/− NK cells) and calcein AM–stained B16F10 cells (transfected with CD155 siRNA or not) were imaged every 2 min from 1 h to 5 h. Representative images of stable and unstable contacts (magnification, 200 ×; electronically zoomed; scale bar, 15 μm; n = 10 movies of 3 independent experiments per group). (B) Ratios of stable and unstable contacts between cancer cells and NK cells (n = 142, 84, 116, 111, and 146 from the left). (C) Binding rates of cancer cells and wild-type or Cd226^−/− NK cells analyzed by flow cytometry (n = 3, mean ± SEM, *p < 0.01).

Figure 7.

Cd226^−/− NK cells weakly inhibit the lung metastasis of cancer cells in vivo. C57BL/6 mice (n = 6) were injected intravenously with B16F10 cells on day 0 and NK cells on day 2. Lungs were collected on day 14 and the metastatic nodules were counted. Mean ± SEM (A) and representative images are shown (B). Representative images of hematoxylin and eosin staining of lung sections (magnification, × 100) (C). *p < 0.01.