Costimulatory Molecule DNAM-1 Is Essential for Optimal NK Education Post Allogeneic Hematopoietic Stem Cell Transplantation

Background: DNAM-1 (DNAX accessory molecule-1, CD226) is a costimulatory molecule that is constitutively expressed by NK cells and CD8+ T cells. Interaction between DNAM-1 on NK cells and CD8+ T cells with its ligands on target cells triggers cell-mediated cytotoxicity and cytokine production. Previous mouse model had showed that the expression of DNAM-1 is associated with NK education. Moreover, Monika's group found that DNAM-1 serves as an intrinsic, readout-independent marker for NK cell education in health donor. Our previous study had 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 post allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the roles of DNAM-1 in NK education post allo-HSCT were unknown.

Aims: In this study, we have investigated the contribution of DNAM-1 expression to NK education post transplantation.

Methods:We prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore the NK cell dynamic education at days 30, 90 and 180 post-transplant. Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry to evaluate of the phenotype as well as functional recovery of NK cells with different maturation expression levels of NKG2A, KIR, and CD57, as well as of activating receptors (NKp30, NKp46, NKG2D, DNAM-1) and CD25, CD122 as well as CD107a and IFN-gamma on NK cells. To study the correlation between the expression of DNAM-1 and effect of donor and host HLA on NK cell education, the expression of DNAM-1 on single-KIR+ NK cells (exhibiting NKG2A, KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) was compared based on the combination between donor/host HLA and donor inhibitory KIR.

Results:

1. The DNAM-1 expression on single-KIR⁺ NK cell ligated by sole donor HLA, or sole host HLA, or both donor and host HLA is higher compared to those single-KIR⁺ NK cells lacking ligands from donor or host or both. From the donor point of view, when donor only presented C1C1 ligand for KIR2DL2/L3, the MFI of DNAM-1 on single KIR2DL2/L3+ NK cells was significantly higher than KIR2DL1+ NK cells and KIR3DL1+ NK cells at 90day (P<0.0001, P=0.046) and 180day (P<0.0001, P=0.034) post transplantation. However, when donor presented C1C1 ligand for KIR2DL2/L3 and Bw4 ligand for KIR3DL1, the MFI of DNAM-1 on single KIR2DL1+ NK cells was significantly lower than KIR2DL2/l3+ NK cells and KIR3DL1+ NK cells at 90day (P<0.0001, P=0.0002) and 180day (P<0.0001, P<0.0001) post transplantation. There was no difference between single KIR2DL1+ NK cells and single KIR3DL1+ NK cells. When donor expressed all HLA(Bw4C1C2) for KIR2DL2/L3, KIR2DL1, KIR3DL1, there was no difference in the expression of DNAM-1 among three single KIR+ NK cells. No matter from the host point or from the combination of donor and host point of view, the DNAM-1 expression differences were same to from donor point of view.

2. There was a clear hierarchy of DNAM-1 expression among NK populations when both of donors and hosts presenting all HLA (Bw4C1C2) for donor KIRs. NK cells with sole KIR or sole NKG2A exhibited higher DNAM-1 expression compared with NKG2A-KIR- NK cells. NK cells with two or three inhibitory KIRs exhibited higher DNAM-1 expression compared with NKG2A+KIR- NK cells. NK cells with 3 inhibitory KIRs and NKG2A expression exhibited maximum DNAM-1 expression at day 30, 90, 180 post transplantation.

3. Meanwhile, the expression of DNAM-1 on NK cells correlated with different ways of education through NKG2A and KIR. There were two fundamental forms of HLA haplotype -21 HLA-B dimorphism: one preferentially supplying CD94:NKG2A ligands (-21M HLA-B), the other preferentially supplying KIR ligands (-21T HLA-B). The expression of DNAM-1 on NKG2A-KIR2DL1+ NK cells was significantly lower when donor or patient was Bw6C1C1 (M/M) compared with when donor or patient was Bw4C2Cx (T/T). However, the expression of DNAM-1 on NKG2A+KIR- NK cells showed no significant difference when donor or patient was Bw6C1C1 (M/M) compared with when donor or patient was Bw4C2C2 (T/T).

Summary: This study demonstrated that no matter from donor or/and host point of view, DNAM-1 expression contributes to optimal NK cells education post allo-HSCT.