Gradual Increase of FcγRIIIa/CD16a Expression and Shift toward IFN-γ Secretion during Differentiation of CD56dim Natural Killer Cells.

Natural killer (NK) cell effector functions include cytotoxicity and secretion of cytokines such as interferon-γ (IFN-γ). The immature CD56bright subset of human NK cells lacks expression of FcγRIIIa/CD16a, one of the low-affinity immunoglobulin G receptors, or exhibits low-density expression (CD56brightCD16-/dim) and produces IFN-γ in response to cytokine stimulation, whereas the mature CD56dimCD16+ subset is the most cytotoxic one. A further differentiation/maturation of the latter subset according to the gradual loss of NKG2A and/or gain of KIR2DL (CD158a and CD158b) has been demonstrated and the ability to produce IFN-γ in response to activating receptor (AR) co-engagement is gradually acquired during terminal differentiation. In the course of flow cytometry analysis of CD56dim NK cells, we noted a substantial intraindividual heterogeneity of expression of FcγRIIIa. FcγRIIIa is unique among ARs: it does not require the co-engagement of other ARs to induce substantial cytotoxicity or cytokine synthesis in CD56dim cells. We, therefore, investigated whether individual differentiation/maturation of polyclonal CD56dim NK cells defined by expression of NKG2A/KIR2DL is related to FcγRIIIa expression and to the heterogeneity of NK cell responses upon FcγRIIIa engagement. When we analyzed unstimulated CD56dim cells by increasing level of FcγRIIIa expression, we found that the proportion of the more differentiated CD158a,h+ and/or CD158b,j+ cells and that of the less differentiated NKG2A+ cells gradually increased and decreased, respectively. FcγRIIIa engagement by using plate-bound murine anti-CD16 monoclonal antibody (mAb) or rituximab or trastuzumab (two therapeutic mAbs), resulted in donor-dependent partial segregation of IFN-γ-producing and/or degranulating CD56dim cells. Importantly, the proportion of CD158a,h/b,j+ cells and that of NKG2A+ cells was increased and decreased, respectively, IFN-γ-producing cells, whereas these proportions were poorly modified in degranulating cells. Similar results were observed after engagement of ARs by a combination of mAbs targeting NKG2D, NKp30, NKp46, and 2B4. Thus, the gradual increase of FcγRIIIa expression is an important feature of the differentiation/maturation of CD56dim cells and this differentiation/maturation is associated with a shift in functionality toward IFN-γ secretion observed upon both FcγRIIIa-dependent and FcγRIIIa-independent stimulation. The functional heterogeneity related to the differentiation/maturation of CD56dim NK cells could be involved in the variability of the clinical responses observed in patients treated with therapeutic mAbs.

ADAM17-mediated shedding of FcγRIIIA on human NK cells: identification of the cleavage site and relationship with activation.

FcγRIIIA/CD16A, the low-affinity receptor for the IgG Fc portion expressed on human CD56(dim) NK cells and involved in Ab-dependent cell cytotoxicity, is shed upon NK cell activation. We found that recombinant a disintegrin and metalloprotease (ADAM) 17 cleaved the ectodomain of FcγRIIIA/CD16A and a peptide for which the sequence encompasses aa 191-201 of the FcγRIIIA/CD16A stalk region but not ADAM10. MALDI-TOF analysis revealed that the peptide was cleaved between Ala(195) and Val(196) (i.e., 1 aa upstream of the expected position). This location of the cleavage site was confirmed by the finding that ADAM17 failed to cleave a peptide in which Ala and Val were reversed. ADAM17 was found to be expressed on NK cells, and stimulation with PMA or N-ethyl-maleimide resulted in the shedding of FcγRIIIA/CD16A and CD62L, a specific substrate of ADAM17. Selective inhibition of ADAM17 prevented the shedding of both molecules. Moreover, the shedding of FcγRIIIA/CD16A was strongly correlated with degranulation when a wide range of CD56(dim) NK cell activating receptors were stimulated, whereas both ADAM17-dependent shedding and internalization were involved in FcγRIIIA/CD16A downmodulation when the latter was engaged. Finally, the shedding of FcγRIIIA/CD16A was restricted to activated cells, suggesting that ADAM17 acts mainly, if not exclusively, in cis. Taken together, our results demonstrated for the first time, to our knowledge, at the molecular level that ADAM17 cleaves the stalk region of FcγRIIIA/CD16A and identified its cleavage site. The shedding of FcγRIIIA/CD16A was at least partially ADAM17 dependent, and it may be considered as a marker of FcγRIIIA/CD16A-independent NK cell activation highly correlated with degranulation.

Fc gamma RIIIa expression is not increased on natural killer cells expressing the Fc gamma RIIIa-158V allotype.

The presence of a valine (V) versus a phenylanaline (F) at position 158 of Fc gamma RIIIa/CD16a improves the affinity for IgG and is associated with higher therapeutic response to rituximab. Increased CD16 expression on natural killer (NK) cells from donors with the VV or VF versus FF genotype has recently been reported. We indeed observed higher binding of the anti-CD16 monoclonal antibody (mAb) 3G8 on NK cells from V carriers (VV = VF > FF). However, the binding of two other anti-CD16 mAbs, LNK16 and DJ130c, decreased with the number of V allele (VV < VF < FF). CD16 transcript levels were independent on the genotype. Rituximab binding to NK cells from V carriers was higher than its binding to FF NK cells at low concentrations (10 and 100 microg/mL). However, the difference was nearly completely abolished at saturating concentrations (>or=1,000 microg/mL). Finally, nearly 100% of CD16-expressing NK cells displayed a complete down-modulation of the receptor after optimal engagement by plate-bound 3G8, whatever the genotype. By contrast, the percentages of NK cells down-modulating CD16 after competitive engagement of the receptor by plate-bound rituximab increased with the number of V allele (FF, 18.2 +/- 8.6%; VF, 32.0 +/- 4.9%; and VV, 42.4 +/- 9.9%). These results are in discrepancy with the expected increased competition that would result from an increased expression of CD16 on VV and VF NK cells. We conclude that increased binding and functional and clinical responses associated with the high-affinity Fc gamma RIIIa-158V are unrelated to an increased expression of this allotype.