Increased Group B Killer Cell Immunoglobulin-Like Receptor (KIR) Haplotypes with Mismatched MHC Class I and Altered NK Repertoire Distribution in Bone Marrow Failure Syndromes

Killer immunoglobulin like receptors (KIR) are expressed on NK cells and a subset of T cells where they bind HLA-C alleles to discriminate self from non-self during anti-tumor and anti-viral responses. KIR receptors exist in inhibitory and activating forms (KIRDL and KIRDS, respectively) that regulate effector functions. The KIR family consists of 14 genes inherited independently from MHC-class I ligands. An imbalance of KIR receptors tilted in favor of more activating KIR genotypes is associated with improved antiviral responses, but also an increase in autoimmunity. We reported previously that KIR/HLA mismatches occur at a higher frequency in bone marrow failure: Aplastic Anemia (AA), Myelodysplastic Syndrome (MDS), Large Granular Lymphocyte leukemia (LGLL), and Paroxysmal Nocturnal Hemaglobinuria (PNH) (Epling-Burnette et al, Blood (ASH Annual Meeting abstracts 2008 112: abstract 4121). We now present data on a larger cohort of patients along with phenotype analysis and individual KIR genotype analysis.

KIR genotyping by bead array (One Lambda, Inc) was performed on 205 cases and resolved in 199 (97%). Samples from 93 unrelated individuals were used as controls. HLA genotyping was analyzed in 190 and 51 cases and controls, respectively. The diagnosis of MDS (n=72), AA (n=59), LGLL (n=46), and PNH (n=28) were confirmed by pathology review. Flow cytometry for KIR2DS1/DL1 was assessed by staining for CD158a (KIR2DS1/DL1) and CD158b (KIR2DS2/DL2/3) on (CD56+/CD16+) NK and (CD4/CD8/CD3) T cells by multiparameter flow cytometry. Antigens at the HLA-C locus were divided into two groups, the HLA-C1 with alleles encoding Ser-77-Asn80 and HLA-C2 alleles encoding Asn-77-Lys-80 that bind KIR2D2 and KIR2D1, respectively. A mismatch was defined as the presence of the KIR gene in the absence of its cognate ligand.

KIR2DL2 and KIR2DS2 gene frequency was increased in the combined BMF group (120/198, 60% DL2 and 117/199, 59%, DS2) compared to controls (43/93, 46% for both DL2 and DS2) (p=0.02 and p=0.058, respectively) in association with a higher frequency of C1 activating and inhibitory receptor mismatches in cases (20 of 190 cases vs. 0 of 51 controls, p=0.009). KIR genotypes can be separated into Group A and B haplotypes with Group A haplotypes containing only one activating KIR (KIR2DS4) and group B haplotypes containing two or more activating KIR. Compared to controls patients with BMF had significantly decreased group A (23%) and increased group B (77%) haplotypes (p0.04). We detected 31 different genotypes in BMF patients and 23 in controls representing both the A and B haplotypes. AA1 was the most common group A genotype in cases (32/35, 91.4%) and controls (49/50, 98%). Specific genotypes that contain KIR2DL2 and DS2 were overrepresented in the BMF cases with AB9 increased in AA (p=0.0054) and AB1 increased in LGLL (p=0.02). KIR phenotyping was performed on NK cells and T cells from 49 BMF patients and 15 controls. KIRs were variably expressed on T cells, and NKG2D had normal expression on NK and T cell populations. CD158a (KIR2DL1/DS1) positive NK cells were significantly reduced in BMF cases compared to controls (17% vs 35%, p0.00008), which was individually significant in AA (18%, p0.003), MDS (20%, p0.01), and LGLL (14%, p=0.0002). The percentage of CD158b (KIR2DS2/DL2/3) positive NK cells, which recognizes the receptors associated with the C1 mismatch, were also significantly reduced in cases compared to controls (30% vs. 43%, p0.02) indicating altered NK repertoire distribution.

KIRs and HLA are inherited independently and have a high level of diversity, but previous results suggest that there is an epistatic relationship between these loci because matched KIR/HLA combinations have evolved through selection. In BMF syndromes, increased frequency of KIR2DL2/KIR2DS2 genotypes in the absence of the appropriate HLA C allele may alter NK and T cell effector responses in favor of autoimmunity. Two to 8 different KIR receptors are present on individual NK cells and these are expressed in a stochastic manner. In previous studies, the expression of a receptor did not depend on the presence of the cognate HLA. Our phenotyping data suggests that the genetic disparities or disease characteristics in BMF may influence NK repertoire distribution leading to fewer KIR positive NK cells with possible pathological consequences.

No relevant conflicts of interest to declare.