Figure 1.
DN NK cells are present within the TINK-cell population.
(A) Representative flow cytometry analysis of the expression of CD27 and CD11b on gated CD56+CD3− TINK cells as compared with pNK cells from autologous patients and healthy control subjects. Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter versus side scatter and an NK-cell gate identifying CD56+CD3− cells. Quadrants depicted were set on isotype controls. (B) The frequency of DN NK cells within the TINK-cell population as compared with that within the pNK-cell populations from autologous patients and healthy control subjects (n = 35; mean±SEM). (C) The frequency of CD11b+SP NK cells within the TINK-cell population as compared with that within the pNK-cell populations from autologous patients and healthy control subjects (n = 35; mean±SEM).
Figure 2.
TINK cells display an immature phenotype.
(A) Representative flow cytometry analysis of the expression of NK-cell maturation receptors (CD57, CD127, CD117 and NKG2A) on gated CD56+CD3− TINK cells as compared with that on pNK cells from autologous patients and healthy control subjects. Quadrants depicted were set on isotype controls. (B) The frequency of CD57+, CD127+, CD117+ and NKG2A+ NK cells within the above-mentioned three NK-cell populations (n = 15; mean±SEM).
Figure 3.
TINK cells display an inactive phenotype.
(A) Representative flow cytometry analysis of the expression of NK-cell activation receptors (CD16, CD226 and NKp30) on gated CD56+CD3− TINK cells as compared with that on pNK cells from autologous patients and healthy control subjects. Quadrants depicted were set on isotype controls. (B) The frequency of CD16+, CD226+ and NKp30+ NK cells within the above-mentioned three NK-cell populations (n = 15; mean±SEM).
Figure 4.
The DN NK subset in TINK cells accounts for the immature phenotype.
(A) Representative flow cytometry analysis of the expression of various surface molecules (CD16, CD57, CD127, CD117, CD226 and NKp30) on the DN NK subset versus the CD11b+SP NK subset in TINK cells. Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter versus side scatter and an NK-cell gate identifying CD56+CD3− cells. DN NK cells were identified based on gating for CD56+CD3−CD11b−CD27−, while CD11b+SP NK cells were identified based on a CD56+CD3−CD11b+CD27− gate. Quadrants depicted were set on isotype controls. (B) The major phenotypic differences detected in the two subsets (DN and CD11b+SP NK) are summarised. Data shown represent the findings from 15 patients (mean±SD).
Figure 5.
DN NK cells have proliferative capacity and poor cytotoxic capacity.
(A and C) Two representative flow cytometry analyses of the expression of Annexin-V and 7-AAD (green graphs) relative to isotype-matched controls (red graphs) on the gated DN NK subset versus the CD11b+SP NK subset in TINK cells. (B and D) The frequency of Annexin-V+ and 7-AAD+ NK cells on two gated NK-cell subsets (n = 6; mean±SD). (E) Two representative flow cytometry analyses of Ki-67 expression (green graphs) relative to isotype-matched controls (red graphs) on the gated DN NK subset versus the CD11b+SP NK subset within TINK cells. (F) The frequency of Ki-67+ NK cells on two gated NK-cell subsets (n = 6; mean±SD). (G) Two representative flow cytometry analyses of CD107a expression (green graphs) relative to isotype-matched controls (red graphs) on the gated DN NK subset versus the CD11b+SP NK subset in TINK cells. (H) The frequency of CD107a+ NK cells on two gated NK-cell subsets (n = 6; mean±SD).
Figure 6.
The frequency of tumour-infiltrating DN NK cells is highly associated with clinical outcome.
(A) Representative flow cytometry analysis of the expression of CD27 and CD11b on TINK cells from NSCLC patients with tumours of distinct stage classifications based on tumour node metastasis (TNM). Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter and side scatter and an NK-cell gate (CD56+CD3−). DN NK cells were analysed by gating on CD56+CD3−CD11b−CD27− cells. Quadrants depicted were set on isotype controls. (B) The frequency of tumour-infiltrating DN NK cells is correlated with the malignant progression of lung carcinoma (n = 20; mean±SEM). (C) The frequency of DN NK cells within tumours is directly correlated with tumour size. The y-axis represents the maximum diameter of the resected tumours.
Figure 7.
The kinetics of DN NK-cell accumulation in tumours is associated with tumour progression in vivo.
(A) To establish tumours, C57BL/6 mice were intrapleurally injected with 5×105 live Lewis lung cancer (LLC) cells. Representative flow cytometry analysis of lung TINK cells at different time-points after LLC injection. Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter versus side scatter and an NK-cell gate using NK1.1+CD3− cells. Quadrants depicted were set on isotype controls. (B) Representative flow cytometry analysis of CD27/CD11b expression in lung TINK cells at different time-points after LLC injection. Quadrants depicted were set on isotype controls. (C) The frequency of TINK cells in lung tissue isolated from C57BL/6 mice at each time-point (n = 6 each). (D–F) The frequency of each subset of lung TINK cells isolated from C57BL/6 mice at each time-point (n = 6 each). All experiments were performed three times with similar results.