Donor and Host Coexpressing KIR Ligands Promote NK Education Post Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Yu Xingxing , Zhao Xiang-Yu , Xu Zhengli , Cao Xunhong , Huo Mingrui , Zhao Xiao-Su , Chang Ying-Jun , Wang Yu , Zhang Xiaohui , Xu Lanping , Liu Kaiyan , Huang Xiaojun 
Published: November 2019

Background:Educated NK cells prevent autoreactive behavior but also permit cytotoxicity against target cells that have down-regulated HLA class I expression. When and how the process of education occurs has not been clearly discerned. Several groups reported that both the donor and host MHC could influence NK cell education in mouse models. In humans, Dulphy et al demonstrated that NK-cell education is shaped by donor HLA genotype. Moreover, our previous study found NK-cell education was shaped by host HLA genotype post allo-HSCT. However, due to the lack of single-KIR+ NK cells, functional analysis limited the full evaluation of the interaction between donor/host HLA and donor inhibitory KIR, so the contribution of donor HLA could not be excluded.
Aims: In this research, we have investigated the relative contributions of donor and recipient HLA to NK cells education, the interplay between functional reconstitution and the involvement of donor/host HLA interaction in NK cell control of leukemia cells.
Methods: Two cohorts of patients were enrolled in this study. We first prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore NK cell phenotypes and functional reconstitution. From June 2012 to April 2016, 276 AML/MDS patients that underwent haploidentical transplantation were enrolled in the second cohort to analyze the effect of donor-host KIR-HLA combinations on relapse post transplantation. Molecular HLA typing and KIR genotyping were performed according to the manufacturer's instructions (One Lambda, Canoga Park, CA, USA). Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry. The cytotoxicity and cytokine secretion of NK cells was determined using CD107a expression and IFN-γ production against the K562 cell line. Single-KIR+ NK cells were grouped into the following groups: (A) nsKIR: where both hosts and donors lacked HLA ligands for one donor KIR; (B) d-rsKIR, where donors and hosts, encoded HLA ligands for donor KIRs; (C) dsKIR, where donors, but not hosts, encoded HLA ligands for donor KIR; and (D) rsKIR, where hosts, but not donors, encoded HLA ligands for donor KIR.
Results:
1. Donor KIR ligated by both donor and host HLA is associated with better single-KIR+ NK cell education among the same patients. KIR2DL2/L3 single+ NK cell exhibited higher reactivity compared to KIR2DL1 single+ NK cell in pairs of donors C1C1 or C1C2 and host C1C1. KIR2DL2/L3 single+ NK cell exhibited higher reactivity than KIR3DL1 single+ NK cell in pairs of donors Bw4C1Cx and host C1Cx. KIR2DL1 single+ NK cell exhibited comparable reactivity with KIR2DL2/L3 single+ NK cell in pairs of donor Bw4C1C2 and host Bw4C1C2.
2. Donor KIR ligated by both donor and host HLA contribute to better single-KIR+ NK cell education among the same single-KIR+ NK cells. KIR2DL2/L3 single+ NK cell in the group of d-rsKIR (C1Cx-C1Cx) exhibit higher reactivity compared with other groups (dsKIR (C1Cx-C2C2), rsKIR (C2C2- C1Cx)). KIR2DL1 single+ NK cells in group of d-rsKIR (C2Cx-C2Cx) exhibited higher reactivity compared with other groups (nsKIR (C1C1-C1C1), dsKIR (C2Cx-C1C1), rsKIR (C1C1-C2Cx)). KIR3DL1 single+ NK cells in groups of d-rsKIR (Bw4Bwx-Bw4Bwx) exhibited higher reactivity compared with other groups (nsKIR (Bw6Bw6-Bw6Bw6), dsKIR (Bw4Bwx-Bw6Bw6), rsKIR (Bw6Bw6-Bw4Bwx)).
3. Both donor and host HLA must coexist for maximum education of NK cells given donor 3 inhibitory KIRs. When both of donor and host presenting all HLA (Bw4C1C2), we showed a remarkable hierarchy of responses among NK populations. NK cells with two inhibitory KIRs for self-HLA exhibited higher NK responsiveness (CD107α and IFN-γ) compared with single KIR+ NK cells. NK cells with 3 inhibitory KIRs for self-HLA exhibited maximum responsiveness.
4. Both donor and host exhibiting all HLA (Bw4C1C2) for donor 3 inhibitory KIRs contributes to least relapse following haploidentical allo-HSCT. In the second cohort, the lowest relapse rate was found in d-rsKIR group (n=31, 0%) compared with rsKIR group (n=55 ,0% vs. 10.0±4.9%, P=0.115), dsKIR group (n=33, 0% vs 14.9%±7.0%, P=0.039), or nsKIR group (n=156, 0% vs. 18%±3.5%, P=0.022).
Summary: This study demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients.

Disclosures
No relevant conflicts of interest to declare.