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Unconventional CD56dim/CD16neg NK cells in HSCT
NK cells, whose frequencies in the recipient PB increase over the following weeks (Figure 1C,D). To validate this working hypothesis, we FACS-sorted uCD56dim, cCD56bright and cCD56dim NK cells from healthy donors (Figure 5B), cultured them with IL-15 plus IL-18 and ana- lyzed the phenotype of proliferating cells diluting car- boxyfluorescein succinimidyl ester (CFSE) at several time points. IL-15 was chosen on the basis of its enhanced abil- ity to prime NK cell proliferation and because it is increased significantly in the blood of the recipient receiv- ing allogeneic (including haploidentical) HSCT.25,31,32 Similarly, IL-18 is known to have a deep impact on NK cell activation and effector-functions.33,34 In this regard, our transcriptional data showed that IL-18 receptor-1 (IL-18- R1) is significantly upregulated in uCD56dim NK cells from hHSCT patients compared to cCD56dim NK cells from healthy donors (FCy (log2) > |1|) (Online Supplementary Table S4). In line with our transcriptional data (Figure 4), we first observed that both uCD56dim and cCD56bright NK cells highly proliferate in response to IL-15 and IL-18, while terminally differentiated cCD56dim NK cells do not. The proliferation index of cCD56bright NK cells is signifi- cantly higher after 14 days of culture compared to those of uCD56dim NK cells (Figure 5C and data not shown for
cCD56dim NK cells). Given the differential expression of NKp46 on uCD56dim and cCD56bright NK cells (Figure 3C), we analyzed the surface levels of this NCR to track the fate of these two proliferating subsets. Although cycling at a higher rate, only a minor and not statistically significant fraction of FACS-sorted NKp46pos/cCD56bright NK cells gen- erated NKp46neg-low/uCD56dim NK cells over time. Indeed, the phenotype of proliferating cCD56bright NK cells was similar (range 75-95%) to that of their resting and parental counterparts at day 0. On the other hand, almost all pro- liferating NKp46neg-low/uCD56dim NK cells gave rise to NKp46pos/cCD56bright NK cells after 14 days of culture, and only a minor fraction retained the NKp46neg-low/uCD56dim phenotype. Finally, we observed that neither FACS-sorted NKp46pos/cCD56bright NK cells nor FACS-sorted NKp46neg- low/uCD56dim NK cells were able to generate cCD56dim NK cells (Figure 5D and Figure 6).
uCD56dim cells expanded early after hHSCT are anergic due to high expression of NKG2A
Our phenotypic analyses showed that donor-derived uCD56dim NK cells expanded early after hHSCT patients are fully armed to efficiently kill tumor cell targets, as already demonstrated in healthy donors.18 Surprisingly,
ABC
Figure 6. Proliferating NKp46neg-low/uCD56dim NK cells differentiate in NKp46pos/cCD56bright NK cells. A) Representative example from a HD of flow cytometry dot plots showing the expression of CD56 on fluorescence-activated cell sorting (FACS)-sorted and CFSE-diluting (CFSEdil) cCD56bright (blue) and uCD56dim (red) from a HD after eight days in culture with interleukin (IL)-15 + IL-18. (B) Representative example from the same HD shown in panel A of flow cytometry histograms showing the level of NKp46 expression, expressed as mean fluorescence intensity (MFI), after eight days in culture with IL-15 + IL+18 (MFI) on cCD56bright (blue line) and uCD56dim (red line) natural killer (NK) cells derived either from FACS-sorted cCD56bright NK cells (blue) or from FACS-sorted uCD56dim NK cells (red). Overlaid gray histograms represent the level of NKp46 expression on the relative freshly purified and FACS-sorted parental NK cells. (C) Summary statistical graph showing the kinetic of NKp46pos/cCD56bright (blue) and of NKp46neg-low/uCD56dim (red) NK cell subsets generated from FACS-sorted cCD56bright (upper graph) and uCD56dim (lower graph) from seven healthy donors. No data are available in panels B and C for cCD56dim NK cells, as they were not proliferating in response to IL-15 and IL-18. Data are expressed as means ± S.D. *P<0.05; ***P<0.001.
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