Page 164 - Haematologica August 2018

A. Roberto et al.
alloreactive NK cell effector functions that are required for a positive clinical outcome of allogeneic HSCTs (Figure 8). It has recently been reported the PT/Cy in hHSCT kills all mature and high proliferating NK cells infused in the recipient with the graft. Indeed, NK cells do not express aldehyde dehydrogenase, thus implying that the imma- ture CD62Lpos/NKG2Apos/KIRneg NK cells reconstituting in the recipients derive from donor HSCs.25 Nonetheless, the kinetic and the clinical impact of NK cell subset distribu- tion in this transplant setting are unknown. Even though the absolute numbers of donor-derived NK cells are restored in the recipients few weeks after the transplant, we show herein that the distribution of their subset takes much longer to acquire a pattern similar to that observed in healthy donors.13 In particular, hHSCT recipients either lack, or have very low frequencies, of circulating cCD56dim NK cells soon after the transplant. The lack of the main cytolytic population that normally accounts for up to 90% of all circulating NK cells is compensated by the expansion, starting from the second week after hHSCT of uCD56dim NK cells, that appear before cCD56dim NK cells and that are present at a higher frequency com-
pared to cCD56bright NK cells early after the transplant. These findings prompted us to first hypothesize that uCD56dim NK cells might represent an additional stage of NK cell differentiation preceding the appearance of cCD56bright and cCD56dim cell subsets. By focusing our analyses on the different transcriptional profiles of NK cell subsets from healthy donors, we first highlighted the peculiar signature of the uCD56dim NK cell subset. These data made it possible to understand how recipients are repopulated over time by specific subsets of donor- derived NK cells that are affected by the peculiar lym- phopenic environment early after hHSCT, and follow a kinetic highly impacting the functional outcome of the transplant. Indeed, both our transcriptional profiles and phenotypic analyses in healthy donors and hHSCT recip- ients showed that uCD56dim lymphocytes are bona fide NK cells and not NK cell precursors, are not an artifact of cry- opreservation, and express surface markers of late differ- entiation such as NKG2D and NKp30 as well as lytic gran- ules indicative of a cytotoxic phenotype. Our results are in line with previous studies showing that uCD56dim NK cells in healthy donors, although present at a very low frequen- cy, represent a distinct subset able to efficiently kill tumor cell targets.15-17 It has also been reported that the lack or decreased expression of CD16 on activated and degranu- lating cCD56dim NK cells is mediated by the metallopro- teinase-17 (ADAM17), thus potentially explaining, at least in part, the origin of uCD56dim NK cells.38,39 Considering that mature and highly proliferating NK cells infused with the graft do not survive to the PT/Cy,25 it is highly unlikely that the action of ADAM17 on activated cCD56dim NK cells could alone explain the high frequencies of uCD56dim NK cells early after hHSCT. Indeed, the expansion of this latter circulating NK cell subset has its peak in the first weeks after hHSCT, when cCD56dim NK cells are either undetectable or present at very low frequencies (Figure 1C,D). Moreover, uCD56dim NK cells are characterized by a remarkably high degree of cellular proliferation in response to cytokine activation, are able to generate cCD56bright NK cells, and express a NKp46neg-low phenotype. These functional and phenotypic features do not belong to terminally differentiated cCD56dim NK cells and are neither induced nor mediated by mechanisms associated with
ADAM17 cleavage properties. As a matter of fact, we did not find any difference in the transcriptional levels of ADAM17 between cCD56dim and uCD56dim NK cells puri- fied early after hHSCT (data not shown). Taken together, these data indicate that ADAM17 likely plays a minor role, if any, in the expansion of uCD56dim NK cells early in hHSCT.
In the context of the cytokine storm characterizing the systemic lymphopenic environment early after allogeneic HSCT (including hHSCT), IL-15 certainly plays a key role as it is highly increased in patients’ sera in the first week after the transplant.25,31 Indeed, the incubation in vitro with IL-15 plus IL-18 of FACS-sorted NKp46neg-low/uCD56dim purified from healthy donors induces their proliferation and preferential differentiation into NKp46pos/cCD56bright NK cells over time. Only a minor fraction of proliferating uCD56dim NK cells retains its parental phenotype follow- ing activation. Additionally we found that, although to a lesser and not statistically significant extent, a small frac- tion of highly proliferating FACS-sorted NKp46pos/cCD56bright NK cells generate NKp46neg- low/uCD56dim NK cells. While these results demonstrate that NKp46 represents an additional surface marker that distinguishes uCD56dim NK cells from both cCD56bright and cCD56dim NK cells, they leave unanswered the question regarding the origin of uCD56dim NK cells. Nonetheless, they indicate the presence of a bi-directional differentia- tion between this latter subset and cCD56bright NK cells in an ex vivo human setting mimicking a lymphopenic envi- ronment highly enriched with IL-15. We are certainly aware that this methodological approach does not resem- ble the complex cellular and molecular interactions occur- ring in the human BM niche during lymphopoiesis. Indeed, neither uCD56dim nor cCD56bright NK cells were able to generate terminally-differentiated cCD56dim NK cells, a process that requires the presence of additional signals delivered by fibroblasts, mesenchymal and stromal cells.40- 42 However, herein we clearly show that both proliferating uCD56dim and cCD56bright NK cells can generate either themselves or their “neighbor” NK cell subset. In this con- text, the high serum level of IL-15 soon after hHSCT25 could also be manipulated to boost a more potent anti- tumor response by cCD56bright NK cells, as recently demon- strated in multiple myeloma.43 Further studies are needed in order to disclose the mechanisms that finely tune the bi-directional transition between uCD56dim and cCD56bright, both under physiologic conditions and in the lym- phopenic setting following allogeneic HSCTs.
Herein, we also demonstrate that the transcriptional profile of uCD56dim NK cells expanded early after hHSCT is distinct from that of their counterparts in healthy donors. This is not surprising in the context of an allogene- ic transplant where different stimuli, such as lymphope- nia, alloreactivity, a high serum level of cytokines, antigen stimulation, opportunistic viral infections and acute GvHD highly influences the quantity and the quality of IR.1,24,25 Indeed, this peculiar systemic environment induces the preferential expansion starting from the sec- ond week after hHSCT of uCD56dim NK cells which, although showing a cytotoxic phenotype, are highly defective in the clearance of tumor cell targets. This cellu- lar functional exhaustion is associated, at least in part, with the transient expression of CD94/NKG2A on all uCD56dim NK cells that account for the majority of the NK cell population within the first weeks following trans-
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haematologica | 2018; 103(8)