Overexpression of KIR inhibitory ligands (HLA-I) determines that immunosurveillance of myeloma depends on diverse and strong NK cell licensing

Abstract

Missing self recognition makes cancer sensitive to natural killer cell (NKc) reactivity. However, this model disregards the NKc licensing effect, which highly increases NKc reactivity through interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) with their cognate HLA-I ligands. The influence of iKIR/HLA-ligand (HLA-C1/C2) licensing interactions on the susceptibility to and progression of plasma cell (PC) dyscrasias was evaluated in 164 Caucasian patients and 286 controls. Compared to controls, myeloma accumulates KIR2DL1^-L2⁺L3^- genotypes (2.8% vs. 13.2%, p < 0.01, OR = 5.29) and less diverse peripheral repertoires of NKc clones. Less diverse and weaker-affinity repertoires of iKIR2D/HLA-C licensing interactions increased myeloma susceptibility. Thus, the complete absence of conventional iKIR2D/HLA-C licensing interactions (KIR2DL1^-L2⁺L3^-/C2C2, 2.56% vs. 0.35%; p < 0.05; OR = 15.014), single-KIR2DL3⁺/C1⁺ (20.51% vs. 10.84%; p < 0.05; OR = 2.795) and single-KIR2DL2⁺/C1⁺ (12.82% vs. 4.9%; p < 0.01; OR = 5.18) interactions were over-represented in myeloma, compared to controls. Additionally, KIR2DL1^-L2⁺L3^- (20% vs. 83%, p < 0.00001) as well as KIR3DL1^- (23% vs. 82%, p < 0.00001) genotypes had a dramatic negative impact on the 3-y progression-free survival (PFS), particularly in patients with low-tumor burden. Remarkably, myeloma-PCs, compared to K562 and other hematological cancers, showed substantial over-expression of HLA-I ("increasing-self" instead of missing-self), including HLA-C, and mild expression of ligands for NKc activating receptors (aRec) CD112, CD155, ULBP-1 and MICA/B, which apparently renders myeloma-PCs susceptible to lysis mainly by licensed NKc. KIR2DL1^-L2⁺L3^-/C2C2 patients (with no conventional iKIR2D/HLA-C licensing interactions) lyse K562 but barely lyse myeloma-PCs (4% vs. 15%; p < 0.05, compared to controls). These results support a model where immunosurveillance of no-missing-self cancers, e.g., myeloma, mainly depends on NKc licensing.

Keywords: HLA-C; HLA-I; KIR; KIR ligands; NK cell activating receptors; NK cell licensing; missing self; myeloma.

Figure 1.

NK cell repertoires in peripheral blood of controls and patients. (A) Analysis of KIR2D expression in peripheral blood NK and T cells. Gates to identify total lymphocyte (a), as well as CD3⁺ (b), CD4⁺ and CD8⁺ (c), CD16⁺CD56⁺ (d), KIR2DS1⁺, L1⁺ and L1⁺S1⁺ (e), and KIR2DL2/S2⁺ and L3⁺ (f) subsets were set in appropriate dotplots. These gates were logically combined to assigned KIR2D expression to CD3⁺CD4⁺, CD3⁺CD8⁺ and CD3⁻CD16⁺CD56⁺ (NK) lymphocytes in a representative patient with the KIR2DL1⁺S1⁺L2/S2⁺L3⁺ genotype. (B) Special analyses were implemented for patients with KIR2DL3 alleles that were non-reactive with anti-KIR2DL3 antibody clone 180701 (presumably KIR2DL3*005 or *015 alleles). As described, KIR2DL3*005 was cross-reactive with anti-KIR2DL1/S1 antibody clone EB6 but not with KIR2DL1 antibody clone 143211. In these patients, the possibility of three genetic backgrounds required cytometric analysis to be adapted: (1) for KIR2DL2⁻ individuals (a, b and c; n = 13) all cells stained with CD158b/j (clone GL183) were assigned as L3⁺ (yellow and orange), disregarding alleged-alleles *005 and/or *015 were homozygous (a, n = 3 ), or heterozygous with others KIR2DL3 alleles (orange) which are reactive with the CD158b antibody (b, n = 5 and c, n = 5); (2) for KIR2DL2⁺ individuals (d and e; n = 6) cells co-reacting with anti-CD158b/j and CD158a/h antibodies were assigned as L3⁺ (yellow), and cells that were non-reactive with CD158a/h antibody as L2⁺ (dark blue); (3) for KIR2DS1⁺/KIR2DL3*005 individuals (c and e; n = 6 ), S1⁺ cells were considered only as those that reacted with CD158a/h but not with CD158a nor CD158bj antibodies (violet and red, in c and e, respectively). (C) NK cell repertoires in peripheral blood of controls and patients. Left, no differences in the number (cells/μL) of total NK cells (CD3⁻CD16⁺CD56⁺) or NK cells expressing KIR2D receptors (KIR2D⁺) or not (KIR2D⁻) between controls and MGUS or myeloma (SMM+MM) patients were detected by using CD158a/h⁺CD158bj antibodies. Right, distribution of peripheral blood NK cell subsets (percent of total NK cells) expressing KIR2DL1, and/or KIR2DL2/S2 and/or KIR2DL3, in total patients (MGUS+SMM+MM) with different KIR2D genotypes. KIR2DL1⁻L2⁺L3⁻ showed only KIR2DL⁻ or KIR2DL2⁺ clones.

Figure 2.

The impact of KIR genotype on myeloma progression free survival depends on tumor burden. (A) Patients with KIR2DL1⁻ (KIR2DL1⁻L2⁺L3⁻, p < 0.01; Pc p < 0.05; top, left graphs) or KIR3DL1⁻ (p < 0.001; Pc < 0.01; bottom, left graphs) genotypes showed significant lower progression free survival than the remaining patients. These differences were more intense and significant (p < 0.00001; Pc p < 0.0001 for both genotypes, middle graphs) in patients with low-tumor burden (aberrant plasma cells -PCs- <95%, equivalent to 6.4 ± 0.8% of PCs in the bone marrow). No differences in the progression free survival were observed in patients with high-tumor burden (aberrant PCs > 95%, equivalent to 29.3% ± 2.7% of PCs in the bone marrow; right graphs). (B) Multivariate logistic regression analysis confirmed that age (OR, 1.04; p = 0.022) and high-tumor burden (OR, 7.84; p = 0.000) significantly and independently increased the risk of myeloma progression. However, presence of any KIR2DL1 (OR, 0.178; p = 0.014) or KIR3DL1 (OR, 0.164; p = 0.016) significantly and independently protected from disease progression.

Figure 3.

Increased expression of HLA-I (“Increasing self”) determines that myeloma plasma cells lysis mainly depends on NK cell licensing. (A) Expression of total-HLA-I (W6/32 antibody, left column) and HLA-C (DT9 antibody, right column) was evaluated in K562 cell line and in residual normal bone marrow lymphocytes (gray line) and tumor cells (bold black line) from T acute lymphoblastic leukemia (T-ALL), acute myeloblastic leukemia (AML) and MM patients. Cells were also stained with isotype control (thin line). K562 and T-ALL blasts completely or partially lost expression of total-HLA-I and HLA-C; AML blasts conserved normal expression of both molecules, but myeloma PCs showed an increased expression of both total-HLA-I and HLA-C compared to normal lymphocytes. (B) Plasma cells from myeloma patients (black bars, n = 15) showed higher expression (Mean fluorescence intensity) of total-HLA-I and HLA-C as well as ligands for activating receptors CD112 and CD155 than PCs from normal donors (striped bars, n = 10) and normal residual lymphocytes (gray bars, n = 15). K562 (white bars) showed much lower expression of HLA-I and HLA-C, and much higher expression of activating ligands than myeloma PCs. **, indicates p < 0.01 in the DMS post-hoc analysis from the ANOVA comparing both normal PCs and residual lymphocytes with myeloma PCs. (C) Ex vivo, un-stimulated purified NK cells from three individuals with variable number of iKIR2D/HLA-C licensing interactions (Controls, white bars and symbols) and three patients with no conventional iKIR2D/HLA-C licensing interactions (KIR2DL1⁻L2⁺L3⁻/C2C2, black bars and symbols) showed similar degranulation and cytotoxic capacity against K562 cell line. However, patients with no conventional iKIR2D/HLA-C licensing interactions showed lower degranulation (p < 0.05) and cytotoxicity (p < 0.05) activity against myeloma PCs (n = 3), cryopreserved from two C2C2 and one C1C1 MM patients. Data represent mean ± SEM of three independent experiments performed for every patient or control against every myeloma PC target. NK cells from controls showed at least one iKIR2DL licensing interaction either for C1C1 or C2C2 myeloma targets.

Figure 4.

Proposed model to explain cytolytic activity of licensed and unlicensed NK cells against normal-self (top), missing-self (medium) and increasing-self (bottom) targets. Our hypothesis sustains that licensed NK cells (left) responding through activating receptors (aRec) can kill missing-self and increasing-self tumors expressing ligands for aRec (aLig), but not normal cells. By contrast, unlicensed NK cells (right) would kill missing-self tumors, but not normal tissue or increasing-self tumors with a mild expression of aLig such as myeloma.