Page 155 - Haematologica August 2018

Unconventional CD56dim/CD16neg NK cells in HSCT
BMT pushed the optimization of different haploidentical HSC transplants (hHSCT) that combined different condi- tioned regimes and immune-modulation therapies.1 Both myeloablative (MAC) and non-MAC (NMAC) T cell- replete (TCRe) hHSCT followed by post-transplant cyclophosphamide (Cy) gave remarkable positive clinical outcomes.2-4
Donor-derived immune-reconstitution (IR) is the most important player ruling out either a positive or negative clinical outcome of allogeneic HSCT.5 Natural Killer (NK) cells are key for the prognosis of allogeneic BMT given their ability to kill viral-infected or tumor-transformed cells in the absence of a prior sensitization to specific antigens.6-8 NK cell recognition of “self” relies on a large family of inhibitory NK cell receptors (iNKRs) including killer cell immunoglobulin-like receptors (KIRs) and C- type lectins, such as CD94/NKG2A, which specifically bind different alleles of major histocompatibility com- plex of class I (MHC-I). A decreased expression or lack of self-MHC-I on target cells unleash NK cell killing via the engagement of several activating NK cell receptors (aNKRs) (i.e., missing self hypothesis).9-11 In the context of allogeneic and non-myeloablative BMT, the presence of a “mismatch” between iNKRs and HLA alleles on recipient cells induces a condition of alloreactivity that makes it possible for donor-derived NK cells to: i) elimi- nate recipient immune cells that survived the condition- ing regimens (i.e., prevent graft reject), ii) kill recipient antigen presenting cells (APCs) presenting host antigens to donor T cells (i.e., avoid the onset of graft-versus-host disease [GvHD]), and iii) clear residual malignant cells in the recipient (i.e., induce graft-versus-leukemia [GvL] effect). These NK cell-mediated effector-functions in mismatched settings are being currently used to develop adoptive NK cell transfer therapies to cure solid and hematologic tumors.7,12
Circulating NK cell subsets are normally defined on the basis of CD56 and CD16 surface expression. Conventional CD56bright/CD16neg-low (cCD56bright) NK cells account for up to 10-15% of the total NK cell population, and are able to secrete a high amount of pro-inflammatory cytokines while displaying poor cytotoxicity. The conven- tional CD56dim/CD16pos (cCD56dim) phenotype identifies another highly cytotoxic subset that comprises up to 90% of NK cells in the bloodstream.13 Other than the patholog- ic NK subsets expanded in immunological disorders and in response to pathogens,14 an additional population of unconventional CD56dim/CD16neg (uCD56dim) NK cells has been identified recently.15-17 Despite being rarely represent- ed in healthy donors, uCD56dim is highly cytotoxic and able to efficiently kill hematologic tumors in vitro.18 A subset of CD56low/CD16low NK cells resembling the phenotype and functions of uCD56dim NK cells is expanded in the bone marrow (BM) of healthy pediatric donors and leukemic patients who have undergone a/b T cell-depleted hHSCT.18,19
The study herein demonstrates that the NK cell IR in TCRe -NMAC-PT/Cy hHSCT with reduced intensity conditioning (RIC) are characterized by the early expan- sion of donor-derived and anergic uCD56dim NK cells showing a peculiar NKG2Apos/NKp46neg-low phenotype. We also demonstrate herein that the blocking of the NKG2A inhibitory pathway on this subset represents an immunotherapeutic target to improve NK cell alloreactiv- ity early after hHSCT.
Methods
Study Design
30 patients were treated according to our published hHSCT protocol approved by the institutional review boards (IRB) of Humanitas Research Hospital.20 Patients and donors signed con- sent forms in accordance with the Declaration of Helsinki. Patients’ clinical features, enrolment criteria and timing of speci- men collections are shown in the Online Supplementary Table S1.21,22 Peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients were isolated as previously described.22,23
Flow cytometry and cell sorting
Cells were thawed in medium (Roswell Park Memorial Institute [RPMI]-1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-glutamine) containing ben- zonase nuclease (Sigma-Aldrich). Cells were stained for 15 min- utes at room temperature (RT) with live/dead fixable dead cell stain kit (Life Technologies) and 20 minutes at RT with fluores- cent-conjugated monoclonal antibodies (mAbs). All mAbs are list- ed in Online Supplementary Table S2. For Ki-67, Perforin and Granzyme (GRZ)-B intracellular staining, cells were fixed and per- meabilized using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's protocol. The gating strategy to identify NK cells within total PBMCs is shown in Online Supplementary Figure S1. Samples were acquired on a LSR Fortessa and LSR II flow cytometers or fluorescence-activated cell sorting (FACS)-sorted with FACS Aria III (BD Biosciences). NK cell absolute counts were obtained by calculating the percentage with- in the lymphocyte gate. Additional Methods are included in the Online Supplementary Material
Results
Preferential expansion of uCD56dim NK cells in the first weeks after hHSCT
We identified CD14neg/CD3neg/CD20neg NK cell subsets in peripheral blood (PB) and donor BM by polychromatic flow cytometry on the basis of their CD56 and CD16 sur- face expression within the lymphocyte gate.13 We did not include CD56neg or NKG2Dneg lymphocytes in our gating strategy, as these cells were contaminants lacking the NK cell surface markers NKp46, NKG2A, Perforin and Granzyme-B (Online Supplementary Figure S1). Our results showed that the absolute numbers of circulating NK cells in hHSCT recipients reach levels similar to those of their healthy donors within four to five weeks after hHSCT with a chimerism that is completely donor-derived after 28 days (Figure 1A,B). These findings confirmed that donor-derived NK cells represent the first lymphoid com- partment to immune-reconstitute in allogeneic HSCT prior to T and B cells.6,7,21,22,24,25
We then characterized the dynamic of circulating NK cell subset IR early after hHSCT. Four distinct NK cell pop- ulations were detectable in both healthy donors and patients and their frequencies were remarkably different within the several time points examined. First, we observed that the frequency of cCD56bright NK cells was significantly higher in the recipients compared to healthy donors starting from the third week from the transplant, and returned to similar physiologic levels 11 weeks after hHSCT. Conversely, the percentage of the cCD56dim NK cells was very low, if not undetectable, in the recipients within the same period, and returned similar to that of their related donors five months after the transplant. The
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