CD11b and CD27 reflect distinct population and functional specialization in human natural killer cells

Abstract

The identification of developmental stages in natural killer (NK) cells, especially in human NK cells, has lagged for decades. We characterize four novel populations defined by CD11b and CD27, which can represent the distinct stages of human NK cells from different tissues. Nearly all NK cells from peripheral blood are CD11b(+) CD27(-) populations whereas NK cells from cord blood have CD11b(+) CD27(-) and CD11b(+) CD27(+) populations. Interestingly, we have found large CD11b(-) CD27(-) populations of NK cells from deciduas. We also demonstrate that each population could be characterized by unique functional and phenotypic attributes. CD11b(-) CD27(-) NK cells display an immature phenotype and potential for differentiation. CD11b(-) CD27(+) and CD11b(+) CD27(+) NK cells show the best ability to secrete cytokines. CD11b(+) CD27(-) NK cells exhibit high cytolytic function. We demonstrate that human NK cells at different developmental stages have special functions and describe a new model of human NK cell differentiation.

Figure 1

CD11b and CD27 reflect different developmental stages in natural killer cells from decidua (dNK), cord blood (cNK) and peripheral blood (pNK) mononuclear cells. (a) Representative flow cytometry analysis of the expression of CD27 and CD11b on gated CD56⁺ CD3⁻ dNK of the first trimester, dNK of the term trimester, cNK and pNK. (b) The frequency of each subset in dNK cells of the first trimester (n = 10). (c) The frequency of each subset in dNK cells of the term trimester (n = 8). (d) The frequency of each subset in cNK cells (n = 9) (e). The frequency of each subset in pNK cells (n = 9).

Figure 2

Each of the four subsets are mainly in a CD34⁻ CD117⁻ CD94^+/− phenotype, among which double negative (DN) and CD27⁺ single-positive (SP) subsets are the majority of CD56^bright CD16⁻ NK cells. (a) Representative flow cytometry analysis of CD34 and CD117 expression on gated CD56⁺ CD3⁻ decidual natural killer (dNK) cells of the first trimester, cord blood NK (cNK) cells and peripheral blood NK (pNK) cells. Results are representative of five experiments. (b) Representative flow cytometry analysis of CD94 expression on dNK, cNK, pNK and the four subsets of NK cells. Results are representative of five experiments. (c) Frequency of CD56^bright CD16⁻ NK cells in total cells and in each subset of dNK cells (n = 6). (d) Frequency of CD56^bright CD16⁻ NK cells in total cells and in each subset of cNK cells (n = 4). (e) Frequency of CD56^bright CD16⁻ NK cells in total cells and in each subset of pNK cells (n = 8).

Figure 3

Double-negative natural killer (DN NK) cells show a more immature phenotype compard with the other NK-cell subsets. (a) The percentages of surface molecule expression using the gated CD56⁺ CD3⁻ decidual NK (dNK) cells of the first trimester, cord blood NK (cNK) and peripheral blood NK (pNK) cells (n = 8) for each experiment. (b) Expression of various surface molecules on gated DN dNK cells versus the other three NK subsets. Results are representative of five experiments. Numbers showed the average fluorescence intensity of each surface molecule.

Figure 4

Double-negative natural killer (DN NK) cells have developmental potential. Decidual cells in the term trimester were cultured with interleukin-2 (IL-2; 100 U/ml) and/or IL-15 (10 ng/ml) for 7 or 14 days. (a) Representative flow cytometry analysis of the expression of CD27 and CD11b on IL-2-cultured decidual NK (dNK) cells at each time-point. Dot plots were gated on live NK cells using a lymphocyte gate using forward scatter versus side scatter and an NK-cell gate using CD56⁺ CD3⁻ cells. (b) The frequency of each subset of NK cells at different time-points. Cells were cultured with IL-2. (c) The frequency of each subset of NK cells at different time-points. Cells were cultured with IL-15. (d) The frequency of each NK-cell subset at different time-point. Cells were cultured with IL-2 and IL-15. (e) Gating strategy of sorting DN NK cells. Dot plots were gated on live NK cells using a lymphocyte gate using forward versus side scatter and an NK-cell gate using CD56⁺ CD3⁻ cells. Then DN NK cells were sorted by gating CD56⁺ CD3⁻ CD11b⁻ CD27⁻. The purity of DN NK cells after sorting was > 95%. (f) Representative flow cytometry analysis of the expression of CD27 and CD11b on IL-2-cultured sorted DN NK cells. (g) Representative flow cytometry analysis of the expression of NKG2A on gated IL-2-cultured dNK cells. (h) The frequency of NKG2A⁺NK cells at each time-point under different culture conditions in (b), (c) and (d). Each result is representative of three experiments.

Figure 5

CD27⁺ single-positive (SP) and double-positive (DP) natural killer (NK) cells have greater cytokine secretion. Typical cytokines produced by human decidual NK cells of the first trimester have been analysed in the four subsets defined by CD27 and CD11b. (a) Representative flow cytometry analysis of interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) in different NK-cell subsets. Results are representative of seven experiments. (b) Frequency of IFN-γ-secreting NK cells in different NK-cell subsets (n = 7). (c) Frequency of TNF-α-secreting NK cells in different NK-cell subsets (n = 7).

Figure 6

CD11b⁺ single-positive natural killer (SP NK) cells have the best ability for killing. (a) The frequency of CD16⁺ NK cells among decidual (dNK), cord blood (cNK) and peripheral blood (pNK) NK cells (n = 10) for each NK cell type. (b) Representative flow cytometry analysis of CD16 expression on cells from each subset of dNK, cNK and pNK cells. Results are representative of six experiments. (c) The frequency of CD16⁺ NK cells in different subsets of dNK, cNK and pNK cells (n = 6). (d) Representative flow cytometry analysis of CD27/CD11b expression on gated NK cells and CD107a expression in different NK subsets and in dNK, cNK and pNK cells. Results are representative of five experiments.