Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells

Abstract

The stages of human natural killer (NK) cell differentiation are not well established. Culturing CD34 ؉ progenitors with interleukin 7 (IL-7), IL-15, stem cell factor (SCF), FLT-3L, and murine fetal liver cell line (EL08.1D2), we identified 2 nonoverlapping subsets of differentiating CD56 ؉ cells based on CD117 and CD94 (CD117 high CD94 ؊ and CD117 low/؊ CD94 ؉ cells). Both populations expressed CD161 and NKp44, but differed with respect to NKp30, NKp46, NKG2A, NKG2C, NKG2D, CD8, CD16, and KIR. Only the CD117 low/؊ CD94 ؉ population displayed cytotoxicity and interferon-␥ production. Both populations arose from a single CD34 ؉ CD38 ؊ Lin ؊ cell and their percentages changed over time in a reciprocal fashion, with CD117 high CD94 ؊ cells predominating early and decreasing due to an increase of the CD117 low/؊ CD94 ؉ population. These 2 subsets represent distinct stages of NKcell differentiation, since purified CD117 high IntroductionNatural killer (NK) cells are CD56 ϩ CD3 Ϫ innate immune effector cells that recognize target cells that have undergone cellular stress, such as malignant transformation or viral infection. 1 Individual NK cells display a diverse repertoire of activating and inhibitory receptors, including the killer immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NKp30, NKp44, and NKp46), and c-lectin receptors (NKG2A/ CD94, NKG2C/CD94, NKG2D, and CD161). The summation of activating signals, when not opposed by inhibitory signaling, leads to the release of granzymes and perforin and target cytolysis. 2 NK-cell activation also results in the elaboration of proinflammatory cytokines, including interferon ␥ (IFN-␥), 3,4 stimulating other compartments of the immune system.The stages of human NK-cell development are not well characterized. Since human NK cells can be differentiated from CD34 ϩ hematopoietic progenitor cells (HPCs) in vitro, such stages of differentiation may be elucidated. Early studies showed that interleukin 2 (IL-2) could induce the differentiation of NK cells from CD34 ϩ HPCs. 5,6 More recently, IL-15 has been found to be critical for NK-cell development since IL-15 Ϫ/Ϫ and IL-15R␣ Ϫ/Ϫ mice show a near-complete absence of NK cells. 7,8 Accordingly, culture of human HPCs with IL-15 gives rise to NK cells, 9 but other cytokines, such as IL-3, IL-7, stem cell factor (SCF), and FLT-3L increase the efficiency of in vitro NK-cell differentiation. [10][11][12] Both SCF and FLT-3L induce the expression of IL-15R␣ mRNA in developing progenitors, inducing IL-15 responsiveness. 11 CD34 ϩ cells cultured with cytokines and stromal cells give higher yields of NK cells that more accurately reflect peripheral blood NK cells with respect to KIR expression. [12][13][14][15] To prevent auto-aggression, NK cells express inhibitory receptors that recognize self major histocompatibility complex (MHC) class I. The current paradigm of NK-cell tolerance suggests that every NK cell must express at least one self MHC-specific inhibitory receptor, since this has been demonstrated at both t...

Ex Vivo Generated Natural Killer Cells Acquire Typical Natural Killer Receptors and Display a Cytotoxic Gene Expression Profile Similar to Peripheral Blood Natural Killer Cells

“…This is in line with the notion that acquisition of NKG2A expression is followed by more prominent expression of other markers of mature NK cells [17], indicating that the outgrowing CD56 + NKG2A + NK cell subpopulation is more mature than the majority of the CD56 + NKG2A -NK cells in the culture. This is further confirmed by the surface expression pattern for KIR and CD16, which are almost exclusively expressed on cells displaying the inhibitory NKG2A/CD94 receptor (Fig.…”

“…It has been previously shown that the NKG2A/CD94 receptor is the major inhibitory receptor on developing mouse NK cells [18] and on mature cytotoxic human NK cells differentiated in vitro using a stromal-feeder cell line [17]. In these studies, NKG2A expression was correlated with the acquisition of cytotoxic properties.…”

“…2E) The most prominent relative upregulation in our NK cell culture system was observed for NKG2A. Since NKG2A can be assumed to be important for the functional maturation of NK cells as a major inhibitory receptor [17,18], we analyzed the correlation of NKG2A expression with other NK cell-related genes upregulated during differentiation. For this purpose, we investigated separately CD14 + , CD56 + NKG2A -, and CD56 + NKG2A + cells for the same set of genes displayed in Fig.…”

Fully functional NK cells after unrelated cord blood transplantation

“…28 In addition, NK-receptor acquisition is an orderly process during NK development, where CD94/NKG2A is first expressed, and followed by acquisition of KIR. [28][29][30][31][32] Interestingly, in this study, we showed that following transplantation, NK cells also displayed major phenotypic features associated with maturity. Indeed, we observed that in spite of the persistent overexpression of CD94/NKG2A, the patterns of NK-cell receptor expression during the first year after SCT revealed great similarity with mature NK cells from healthy donors, including major KIRs, NKG2C NKG2D, NKp44, NKp80, ILT2, 2B4 and LAIR1.…”

Donor major histocompatibility complex class I expression determines the outcome of prenatal transplantation

“…NK cells clearly respond to altered class I ligand expression and may represent an innate immune barrier to prenatal transplantation even during human fetal development [13][14][15]. Murine Ly49 receptor expression occurs postnatally and may be responsible for the progressive rejection of the transplanted B6.TAP −/− cells which occurred between 4-6 weeks of age [16][17][18].…”