Resistance of Hematopoietic Stem Cells Lacking the KIR Ligand to Autologous NK Cell Attacks in Patients with Acquired Aplastic Anemia
Authors: Maruyama Hiroyuki , Espinoza Luis J. , Katagiri Takamasa , Zaimoku Yoshitaka , Kashiwase Koichi , Saji Hiroo , Ogawa Seishi , Nakao Shinji 
Published: November 2019

Abstract
Normal blood cells, including hematopoietic stem cells (HSCs), express KIR ligands (KIR-Ls) to protect themselves from an autologous NK cell attack, and malignant cells lacking KIR-Ls elicit NK cell-mediated killing of themselves. This missing-self mechanism is believed to play an important role in the elimination of malignant cells. However, the mechanisms underlying the killing of KIR-L-lacking malignant cells by NK cells remain unclear due to the heterogeneity of tumor cells in terms of their proliferative capacity, and also because other accessory molecules may be involved in the NK cell attacks, in addition to KIR-Ls. This makes it difficult to clarify the interaction between NK cells and KIR-L-lacking target cells. The lack of class I HLA occurs not only in malignant blood cells, but also in the normal leukocytes of patients with acquired aplastic anemia (AA). These HLA-lacking leukocytes, detectable in 13% of patients with AA, are derived from HSCs that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH), and thereby escape the cytotoxic T-cell (CTL) attack against HSCs. The 6pLOH may involve KIR-L loss in some patients, leading to a change in the susceptibility of the affected HSCs to NK cell-mediated killing. Unlike malignant cells, HLA-lacking leukocytes are essentially the same as the wild-type leukocytes, except for the HLA expression. Studying 6pLOH (+) AA patients with leukocytes lacking KIR-Ls should therefore be useful for clarifying the roles of KIR-Ls and other accessory molecules in the target cell killing by NK cells. Screening of 389 patients with AA using flow cytometry and a SNP array analysis revealed that there were HLA-A allele-lacking leukocytes in 60 (15.4%) patients, which included 36 C1/C2 and 24 Bw4/Bw6 heterozygotes. Unexpectedly, a lack of KIR-Ls as a result of 6pUPD was found in five patients (13.9%, C1 missing in two and C2 missing in three) of the 36 C1/C2 heterozygotes and in five (20.8%) of the 24 Bw4/Bw6 heterozygotes, although the proportion of patients lacking a KIR-L-containing haplotype (20.8%) was significantly lower than that of patients lacking a haplotype that did not contain KIR-Ls (79.2%). Moreover, the median percentage of HLA-A-lacking granulocytes in the 10 patients who lacked a KIR-L-containing haplotype (12.4%, 0.44%-50.3%) was significantly lower than that (55.3%, 1.4%-99.4%) in the 26 patients who lacked a haplotype that did not contain KIR-Ls, suggesting that the HSCs lacking KIR-Ls or their progenies are susceptible to autologous NK cells to some extent, but are not eliminated completely. To clarify the mechanisms underlying the HSC resistance to NK cells, we determined the KIR gene repertoire and the haplotype of seven patients whose 6pLOH(+) leukocytes were lacking a KIR-L-containing haplotype. All patients possessed inhibitory KIR genes responsive to corresponding KIR-Ls, a finding that negates the possibility that NK cells failed to undergo licensing in these patients. Although the frequency of the KIR-B haplotype, a haplotype associated with a higher cytotoxic function of NK cells, in the seven patients was lower (14%) than that in Japanese healthy individuals (40.1%), two patients possessed the KIR-B haplotype. Phenotypic analyses of the NK cell subsets defined by anti-2DL1, anti-2DL2/2DL3 and anti-3DL1 antibodies showed that all seven patients had a similar percentage of the eight different NK cell subsets, which included 0.5 to 8% of effector NK cells capable of killing leukocytes lacking corresponding KIR-Ls. The expression level of HLA-E was comparable between HLA-A-lacking and HLA-A-retaining monocytes. The expression of NKG2A on the effector NK cells was also comparable to that of the other NK cell subsets in the 6pLOH(+) patients. Our study demonstrated, for the first time, that HSCs lacking KIR-Ls can evade autologous NK cell attack through an as yet unknown mechanism(s) and can continue to generate blood cells in patients with AA.

Disclosures
No relevant conflicts of interest to declare.