Anti-Leukemia Activity of Alloreactive NK Cells in Haploidentical HSCT in Pediatric Patients: Re-Defining the Role of Activating and Inhibitory KIR
Authors: Pende Daniela , Marcenaro Stefania , Falco Michela , Martini Stefania , Bernardo Maria Ester , Montagna Daniela , Romeo Elisa , Martinetti Myriam , Maccario Rita , Mingari Maria Cristina , Moretta Lorenzo , Locatelli Franco , Moretta Alessandro 
Published: April 2020

Abstract
T-cell depleted hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT) has been reported to benefit from the graft-versus-leukemia effect mediated by natural killer (NK) cells when donor displays NK alloreactivity versus the recipient. NK alloreactivity is mediated by NK receptors, namely Killer Ig-like receptors (KIR) which are specific for allotypic determinants that are shared by different HLA-class I alleles (referred to as KIR ligands). It is known that KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (Bw4 group) and KIR3DL2 recognizes HLA-A3 and –A11 alleles. KIR2D/3DL are inhibitory receptors that, upon engagement with the cognate ligand, inhibit lysis. Activating KIRs, highly homologous in the extracellular domain to the inhibitory counterparts, are KIR2DS1, KIR2DS2 and KIR3DS1, but only KIR2DS1 has been shown to specifically recognize C2 group of alleles expressed on B-EBV cells. We analyzed 21 children with leukemia receiving haplo-HSCT from a relative after a myeloablative conditioning regimen; in all pairs, the expression of a given KIR ligand (HLA class I allele) of the donor was missing in the patient (i.e. KIR ligand-mismatched haplo-HSCT). T-cell depletion was performed through positive selection of CD34+ cells; no pharmacological immune suppression was employed after HSCT. KIR genotype of all donors was evaluated to detect the presence of the various inhibitory and activating KIR genes. Phenotypic analyses were performed on NK cells derived from the donor and the patient at different time points after HSCT. Thanks to the availability of new mAbs able to discriminate between the inhibitory and the activating forms of a certain KIR, we could identify the alloreactive NK cell subset at the population level. These alloreactive NK cells express the KIR specific for the KIR ligand-mismatch (permissive inhibitory KIR) and the activating KIR (if present), while they do not express all inhibitory KIR specific for the patient HLA alleles and NKG2A. Thus, in most instances, we could precisely identify the size of the alloreactive NK cell subset in the donor and in the reconstituted repertoire of the recipient. Functional assays were performed to assess alloreactivity, using appropriate B-EBV cell lines and, if available, patient’s leukemia blasts. In some cases, also NK cell clones were extensively studied, for phenotype and receptor involvement in killing activity. We found that, in most transplanted patients, variable proportions of donor-derived alloreactive NK cells displaying anti-leukemia activity were generated and maintained even at late time-points after transplantation. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying HLA alleles belonging to the C2 specificity. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that co-expressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role for the KIR2DS2 activating receptor in leukemia cell lysis could not be established. Taken together, these findings provide new information on NK alloreactivity in haplo-HSCT that may greatly impact on the selection of the optimal donor.