MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response

Abstract

Variegated expression of 6 inhibitory HLA class I-specific receptors on primary NK cells was studied using high-dimension flow cytometry in 58 humans to understand the structure and function of NK-cell repertoires. Sixty-four subsets expressing all possible receptor com-binations were present in each repertoire, and the frequency of receptor-null cells varied among the donors. Enhancement in missing-self response between NK subsets varied substantially where subset responses were defined by donor KIR/HLA allotypes, reflecting the differences in interaction between inhibitory receptors and their ligands. This contrasted to the enhancement conferred by NKG2A, which was constant and of intermediate strength. We infer a mechanism that modulates frequencies of the NK subsets displaying diverse levels of missing-self response, a system that reduces the presence of KIR-expressing subsets that display either too strong or too weak a response and effectively replaces them with NKG2A-expressing cells in the repertoire. Through this high-resolution analysis of inhibitory receptor expression, 5 types of NK-cell repertoire were defined by their content of NKG2A(+)/NKG2A(-) cells, frequency of receptor-null cells, and degree of KIR receptor coexpression. The analyses provide new perspective on how personalized human NK-cell repertoires are structured.

Figure 1

All combinations of inhibitory MHC class I receptors are represented in human NK-cell repertoires. In the upper part of the figure, the gray bars give the frequencies for CD56^dim NK cells having the receptor combination denoted by the black, filled boxes below each bar; shown are data for a representative donor (rd). The lower part of the figure gives mean (m) and range (r) frequencies for NK subsets expressing different numbers of inhibitory MHC class I receptors, both for the representative donor alone and all 27 donors analyzed. The donors are grouped by KIR haplotype combination. A and B refer to KIR haplotypes defined at the low resolution of gene content; A1 is defined at the high resolution of allele content. The representative donor has the A1A1 genotype. See Figure S1 for donor KIR and HLA class I genotypes.

Figure 2

Diverse NK repertoires are characterized by the degree of receptor coexpression, balance of NKG2A/KIR, and receptor-null cell content. (A) Shown are NK-cell repertoires for 3 KIR A1A1 haplotype donors. Each observed repertoire (■) is compared with a simulated repertoire (□), which is the repertoire expected by the product rule. Donors I and II have Bw4 and C1; donor III has only C1. (B) Comparison of the expression frequencies for the 6 MHC class I receptors expressed independently (single-positive cells) and in combination with KIR. ● represent the frequencies observed for the 58 donors; ○ represent frequencies predicted by the product rule. (C) The inverse correlation between the frequencies of KIR⁺NKG2A⁻ and KIR⁻NKG2A⁺ NK cells in 58 donors with varied KIR and HLA types. The observed frequencies (●) are compared with those expected from the product rule (○). (D) For each donor, the frequency of receptor-null NK cells is plotted against the frequency of cells expressing one or more KIR (▴) and the frequency of cells expressing NKG2A (◇).

Figure 3

Human NK-cell repertoires form 5 broad groups according to inhibitory receptor phenotypes. NK-cell repertoires divide into KIR-dominant (left half of figure) and NKG2A-dominant (right half of figure) repertoires. (A) KIR-dominant repertoires further divide into 3, based on the frequencies of receptor-null cells and KIR-KIR coexpression (●). (B) Whereas frequencies of KIR single expression (○) and KIR-KIR coexpression (●) are similar in type 1 repertoires, KIR coexpression is less frequent in type 2 repertoires and even more so in type 3 repertoires. (C,D) NKG2A-dominant repertoires are further divided according to the frequency of receptor-null cells (C). The difference between type 4 (□) and type 5 (■) repertoires is apparent in panel D where the ratio of NKG2A coexpression with KIR against NKG2A single expression is higher in type 4 than type 5 repertoires. (E) Representative profiles of the 5 repertoire types.

Figure 4

NK subsets display defined levels of enhanced response to “missing-self” that are determined by the combination of KIR and HLA polymorphisms in the donor. (A) NK subsets defined by single inhibitory MHC class I receptors respond differently to 721.221 target cells. For this donor (HLA-C1, Bw4; KIR A1A1), the NK subsets bearing only LILRB1, KIR2DL1, or 3DL2 gave weak responses, such as the receptor-null subset, whereas NK cells bearing NKG2A, KIR2DL3, or 3DL1 gave strong responses. (B) NK subsets, from the same donor, expressing different combinations of KIR3DL1, 2DL3, 2DL1, and NKG2A were analyzed for their cytokine and cytotoxic response to 221 cells. The vertical line gives the response level of the receptor-null subset. (C) How different combinations of HLA class I ligands and NK-cell receptors enhance the missing-self response of NK subsets to 221 cells. In all 58 donors, the enhanced response of NKG2A single-positive cells was constant (dotted vertical line), whereas the effects on KIR single-positive cells varied with donor HLA and KIR genotype. NKG2A, n = 58; 2DL1/S1⁺ C2⁻, n = 42; C2⁺2DL1*004⁺, n = 4; C2⁺2DL1*004⁻, n = 11; 2DL3⁺ Cw*7⁺, n = 16; Cw*7⁻Cw*12⁺, n = 7; Cw*7⁻Cw*12⁻B*46⁺, n = 4; Cw*7⁻Cw*12⁻ Cw*1402⁺, n = 4; Cw*01/03/08/1403⁺, n = 10; 2DL2/3⁺ C2/2, n = 5; 3DL1*001/002/15/20⁺ B*51/52⁺, n = 13; B4403⁺, n = 5; B*13⁺, n = 2; 3DL1⁺Bw4⁻ A*24⁺, n = 13; A*24⁻, n = 9; 3DL1*1502⁺, n = 22; 3DL1*007⁺, n = 5; 3DL1*005⁺, n = 8; 3DS1⁺, n = 1. *P < .05, ** P < .005, *** P < .001. Comparison of the effect by C1 was significant by ANOVA. P < .05 was considered statistically significant.

Figure 5

The enhanced levels of subset response conferred by receptor coexpression are dampened to various degrees by the combinations of HLA allotypes on target cells. (A) The capacity of 221 transfectant expressing C1 (top subpanel) and Bw4 (bottom subpanel) to inhibit NK cells bearing their cognate KIR2DL2/3 and 3DL1*1502 receptors did not differ between NK cells from donors who have or lack the cognate ligand. The C1 inhibitor was Cw*1202, and the Bw4 inhibitor was B*5801. The results are the means obtained from the following donors: KIR2DL2/3⁺ Cw*07⁺, n = 13; Cw*07⁻, n = 19; C1⁻, n = 6; 3DL1*1502⁺ Bw4⁺, n = 15; Bw4⁻, n = 5. (B) Coexpression of inhibitory MHC class I receptors increases the enhanced IFN-γ response to missing-self, but the effect is attenuated in cells with multiple receptors. The response of NK cells expressing different combinations of receptors (2DL3, 3DL1, and NKG2A) was compared among donors who have the identical A1A1 KIR genotype and both the Bw4 and C1 ligands. Donors having Cw*07 (♦, n = 3) are distinguished from donors lacking Cw*07 (◇, n = 4). (C) NK cells expressing inhibitory receptors for 2 different HLA class I ligands are not fully inhibited by either of the ligands alone. NK cells expressing KIR2DL3 and 3DL1 from Bw4⁻ donors are fully inhibited by C1, whereas 2DL3⁺ 3DL1⁺ cells from Bw4⁺ donors are incompletely inhibited by C1 (top subpanel). Similarly, NK cells expressing KIR2DL3 and 3DL1 from Bw4⁻ donors are fully inhibited by Bw4, whereas 2DL3⁺3DL1⁺ cells from Bw4⁺ donors are incompletely inhibited by Bw4 (bottom subpanel). The C1 inhibitor was Cw*1202, the Bw4 inhibitor was Bw*5801, and the 3DL1 allele was 1502. 5 Bw4⁺ donors and 2 Bw4⁻ donors with the A1A1 KIR genotype were studied. Plots show mean values with the range of variation indicated by the SEM. *P < .05, **P < .005. (D) NK subset responses against allogeneic PHA blasts and 721.221. KIR ligands of 3 group A homozygote donors are shown on the left, and HLA genotypes of PHA blasts are shown at the top of the each panel. Receptor combinations of KIR2DL1, 2DL3, 3DL1, or NKG2A on each NK subset are indicated by the presence of a dotted line in the center of each box; gray shading in the left half of each box indicates presence of a ligand conferring enhancement of missing-self response to a subset; gray shading in the right half of each box indicates presence of the ligand on the target cell that induces inhibition. Length of bar indicates missing-self response as assessed by IFN-γ production. Subset frequencies for each donor are indicated in the far left column. Donor HLA allotypes are: donor 1, HLA-B*07/57, Cw*06/07; donor 2, HLA-B*52/58, Cw*03/12; donor 3, HLA-B*39/40, Cw*07/07.

Figure 6

The balance in KIR/NKG2A expression is determined by the number of strong KIR-HLA interactions and results in equilibrium of total NK-cell repertoire response. (A) For the donor panel, the ratio in the frequency of NK cells that lack NKG2A (sum of the null subset and the NKG2A⁻KIR⁺ subset) to NK cells that have NKG2A is plotted against the number of strong ligands. All C2, Cw*07, Cw*12, B*46, and all Bw4⁺ HLA-B allotypes, with the exception of B*13 were considered strong ligands, but to NK cells that have NKG2A in donors having the cognate inhibitory KIR. The mean ratio (2.4) for the panel is given by the horizontal dotted line. Ratios greater than the mean represent repertoires dominated by KIR (KIR-dominant) and ratios below the mean represent repertoires dominated by NKG2A (NKG2A-dominant). The correlation of KIR-dominant repertoires with one ligand and of NKG2A-dominant repertoires with 2 or more ligands was significant. *P < .05. (B) Comparison of the ratio of the expression frequencies of KIR2DL3 and NKG2A with the ratio of their enhanced missing-self responses. Closed symbols represent donors having strong KIR3DL1 and/or 2DL1 responses; open symbols represent donors with weak 3DL1 and 2DL1 responses. Between receptor expression and response, 4 patterns were discerned. On the right side of the panel are donors with a stronger response of 2DL3 over NKG2A and either strong (●) or weak (○) KIR3DL1 and/or 2DL1 responses. On the left side are donors with stronger response of NKG2A over 2DL3 and either strong (▴) or weak (Δ) 3DL1 and 2DL1 responses. (C) The relative proportions of 5 NK subsets in 5 donors (Figure 3E) representing the 5 types of NK-cell repertoire. The type 1 repertoire is characterized by a larger subset of NK cells coexpressing inhibitory KIR, type 2 by receptor-null cells, type 3 by cells expressing single inhibitory KIR, type 4 by cells coexpressing KIR and NKG2A, and type 5 by a higher proportion of single-positive NKG2A cells compared with coexpression of NKG2A with KIR. (D) Overall levels of enhancement in missing-self response of 58 NK-cell repertoires. The donors are grouped according to the 5 repertoire types and plotted according to the sum of the enhanced response for all receptors having a cognate ligand. The type 2 null-dominant repertoires have a significantly lower enhanced response with a mean of 13 compared with 22, 20, 22, and 16 for the type 1, 3, 4, and 5 repertoires, respectively (P < .005). The summed enhanced response for the donors was less than 35 (dotted horizontal line) with one exception. *P < .05. ***P < .005.

Figure 7

NK subset frequencies in human NK-cell repertoires vary in their amount of departure from the product rule. (A) For each donor, the deviation of the observed NK-cell repertoire from the product rule was assessed for 5 parameters: single KIR expression (▴), KIR-KIR coexpression (■), NKG2A expression alone (○), NKG2A-KIR coexpression (▵), and no receptor expression (●). The donors are ordered from left to right according to decreasing deviation in single KIR expression. (B) KIR coexpression is higher than expectation under the product rule regardless of ligand presence. The amount of deviation from expectations under the product rule is shown for NK subsets coexpressing 2 KIR (2DL1-2DL3, 2DL3-3DL1, 3DL1-2DL1). This amount is shown separately by the number of cognate ligands (0-2) for the 2 KIR. The deviations are all positive in these subsets from donors with type 1, 2, 4, and 5 repertoires. The deviations display a different pattern for cells from donors with type 3 repertoires, where KIR coexpression is drastically reduced from frequencies predicted under stochastic coexpression, but only in those subsets that have developed in the presence of autologous cognate ligands. All donors are group A homozygotes. Donors for type 1, 2, 4, and 5 repertoires: n = 24; those for type 3 repertoires: n = 5. (C) KIR coexpression is enhanced when multiple KIR confer similar levels of enhanced missing-self response. The ratio of missing-self response between KIR with the strongest response and that from the second strongest are compared in donors with type 1 (●, n = 7) and type 3 (○, n = 8) repertoires. The plots display an association with the amount of deviation in KIR-KIR coexpression.