NK cell terminal differentiation: correlated stepwise decrease of NKG2A and acquisition of KIRs

Abstract

Background: Terminal differentiation of NK cells is crucial in maintaining broad responsiveness to pathogens and discriminating normal cells from cells in distress. Although it is well established that KIRs, in conjunction with NKG2A, play a major role in the NK cell education that determines whether cells will end up competent or hyporesponsive, the events underlying the differentiation are still debated.

Methodology/principal findings: A combination of complementary approaches to assess the kinetics of the appearance of each subset during development allowed us to obtain new insights into these terminal stages of differentiation, characterising their gene expression profiles at a pan-genomic level, their distinct surface receptor patterns and their prototypic effector functions. The present study supports the hypothesis that CD56dim cells derive from the CD56bright subset and suggests that NK cell responsiveness is determined by persistent inhibitory signals received during their education. We report here the inverse correlation of NKG2A expression with KIR expression and explore whether this correlation bestows functional competence on NK cells. We show that CD56dimNKG2A-KIR+ cells display the most differentiated phenotype associated to their unique ability to respond against HLA-E+ target cells. Importantly, after IL-12+IL-18 stimulation, reacquisition of NKG2A strongly correlates with IFN-gamma production in CD56dimNKG2A- NK cells.

Conclusions/significance: Together, these findings call for the reclassification of mature human NK cells into distinct subsets and support a new model, in which the NK cell differentiation and functional fate are based on a stepwise decrease of NKG2A and acquisition of KIRs.

Figure 1. Phenotype patterns of CD56^bright (Bright), CD56^dimNKG2A⁺(D2A+) and CD56^dimNKG2A⁻(D2A−) NK cell subsets from healthy controls.

Representative samples (left panels) and box and whiskers plots (right panels) are presented for each marker from at least 16 healthy controls. *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 2. Whole genome microarray analysis.

CD56^brightCD16⁻ (Bright), CD56^dimNKG2A⁺ (D2A+) and CD56^dimNKG2A⁻ (D2A−) NK cell subsets from 4 healthy donors (#1, #2, #3 and #4) were sorted and analysed using pan-genomic microarrays. (A) Left panel, Hierarchical clustering analysis of significantly modulated genes. The six clusters were numbered C1 to C6. Right Panel, alternative representation of each cluster showing the relative intensity of genes expression. Median relative intensities are represented by a red line. (B) Gene ontogeny analysis was performed with DAVID software on genes of clusters 2, 4 and 5. Highly significant terms are shown with the associated genes (boxes) and their expression profile in the three NK cell subsets. Median relative intensities are represented by a red line.

Figure 3. Extensive analysis of KIR expression.

Cell surface expression of whole KIRs (A), or single KIR (B) on CD56^bright (open bars), CD56^dimNKG2A⁺ (grey bars) and CD56^dimNKG2A⁻ (black bars) NK cells subsets from 32 healthy controls. *: p<0,05; **: p<0.01; ***: p<0.001.

Figure 4. IFN-γ production is associated with NKG2A re-acquisition.

(A) Intracellular expression of IFN-γ by CD56^bright (Bright), CD56^dimNKG2A⁺ (D2A+) and CD56^dimNKG2A⁻ (D2A−) NK cell subsets from 7 healthy donors, after cell sorting and IL-12/IL-18 overnight activation. *: p<0.05; **: p<0.01; ***: p<0.001. (B) Representative example of NKG2A expression and IFN-γ production, before (UT) and after stimulation with IL-12/IL-18 in CD56^bright (Bright), CD56^dimNKG2A⁺ (D2A+) and CD56^dimNKG2A⁻ (D2A−) NK cell subsets.

Figure 5. The CD56^dimNKG2A⁻KIR⁺ NK cells are the only one able to degranulate against HLA−E+ target cells.

CD107a degranulation assays of CD56^bright (Bright), CD56^dimNKG2A⁺ (D2A+) and CD56^dimNKG2A⁻ (D2A−) NK cell subsets were accessed without target, or against K562 or RAJI cells in the presence of 1 µg of rituximab (ADCC) (A), or against 721.221 target cells that do or do not express HLA-E (LCL-221-AEH) (C). Box and whiskers plots of CD107a degranulation capacities of CD56^bright (Bright), CD56^dimNKG2A⁺KIR− (D2A+KIR−), CD56^dimNKG2A⁺KIR⁺ (D2A+KIR+), CD56^dimNKG2A⁻KIR⁺ (D2A−KIR+) and CD56^dimNKG2A⁻KIR⁻ (D2A−KIR−) NK cell subsets from 11 healthy controls. NK cells were tested without target cells, against RAJI cells in the presence of 1 µg of rituximab (ADCC), or K562 cells (B) or 721.221 target cells that do or do not express HLA-E (D). *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 6. Stepwise decrease of NKG2A and acquisition of KIRs is shown by specific markers of NK cell maturation.

Expression of CD62L, granzyme-K and CD27 (A) or ILT2, CD57, Siglec-9 and FCRL6 (B) on CD56^bright (Bright), CD56^dimNKG2A⁺KIR⁻ (D2A+KIR−), CD56^dimNKG2A⁺KIR⁺ (D2A+KIR+), CD56^dimNKG2A⁻KIR⁺ (D2A−KIR+) and CD56^dimNKG2A⁻KIR⁻ (D2A−KIR−) NK cell subsets from at least 16 healthy donors. *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 7. Model of terminal differentiation of NK cells.

NK cell repertoire gradually changed from CD56^bright towards a CD56^dim dominant response; CD56^dimNKG2A⁺KIR⁻ cells then develop into CD56^dimNKG2A⁺KIR⁺ and then terminally differentiate into CD56^dimNKG2A⁻KIR⁺ cells. Expression of KIR in a few CD56^bright NK cells also suggested that a part of those cells give rise directly to CD56^dimNKG2A⁺KIR⁺. This model also includes the potential re-expression of NKG2A on CD56^dimNKG2A⁻ NK cells after stimulation. Hyporesponsive CD56^dimNKG2A⁻KIR⁻ NK cells are integrated as cells that failed to acquire KIR before NKG2A loss due to their incompletely differentiate phenotype.