Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

Abstract

Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

Figure 1. Assessment of NK cell repertoire diversity based on expression of unique receptor profiles

(A) Frequencies of the 50 most abundant NK cell phenotypes based on expression of 28 receptors for 12 unrelated individuals (HNK-001 through HNK-012) and 5 pairs of monozygotic twins (Twins 1a/1b – 5a/5b). Each column represents a phenotype, with colored boxes indicating receptor presence. (B) Comparative correlation of each twin with his/her identical twin of the frequencies of the 50 phenotypes shown in A. Spearman’s correlation coefficient is displayed. (C) Comparative correlation of each healthy unrelated individual with the other 11 individuals of the frequencies of the 50 phenotypes shown in A.

Figure 2. Expression patterns of inhibitory NK cell receptors in unrelated individuals and monozygotic twins

(A) Inhibitory receptor profile of NK cells in 12 unrelated individuals and 5 pairs of monozygotic twins as in Figure 1A. The analysis was restricted to the evaluation of expression profiles of the six inhibitory receptors KIR2DL1, KIR2DL2/L3/S2, KIR2DL5, KIR3DL1, LILRB1, and NKG2A. (B) Comparative correlation of each twin with his/her identical twin of the frequencies of the 50 phenotypes shown in A. Spearman’s correlation coefficient is displayed. (C) Comparative correlation of each healthy unrelated individual with the other 11 individuals of the frequencies of the 50 phenotypes shown in A.

Figure 3. Clustering analysis of NK cells using SPADE reveals diverse receptor expression patterns

(A) Representative SPADE trees of NKG2A, NKG2D, KIR2DL1 and KIR2DS4 for monozygotic twins 5a (top) and 5b (bottom). Node color represents signal intensity of each marker and size represents frequency. (B) Representative SPADE trees of NKG2A, NKG2D, KIR2DL1 and KIR2DS4 for 2 healthy unrelated individuals, HNK-003 (top) and HNK-007 (bottom).

Figure 4. Identification and phenotypic evaluation of CD57⁺NKG2C⁺ ‘memory-like’ NK cells

(A) Skeleton structures of a SPADE tree from healthy unrelated individuals (HNK) and monozygotic twins (Twin) with NKG2C-expressing nodes highlighted. (B) Representative SPADE trees of the NKG2C-expressing nodes shown in (A) from 5 unrelated individuals. (C) Representative SPADE trees of NKG2C-expressing nodes for 1 pair of monozygotic twins, as in (B).

Figure 5. NK cell receptor co-expression patterns and population structure

(A) Spearman’s rank correlation matrices of co-expression profiles for each receptor pair for all NK cells from all 22 donors. (B) Hierarchical clustering dendrogram of NK cell receptor profiles from all NK cells from all 22 donors. (C) Principal component analysis of receptor expression patterns from all NK cells from all 22 donors. Dots indicate the contributions of each receptor to principal component 1 (x-axis) vs. principal component 2 (y-axis).

Figure 6. Genetics dictate the expression of most single NK cell receptors, but not the combinatorial assortment of phenotypes

(A) Inverse Simpson for the population of cells expressing each single receptor. Boxplots show the median, 25^th and 75^th percentiles, and total range of all 22 donors. Yellow lines show a representative twin pair. Red line indicates an unpaired representative twin. (B) Summary of Intraclass Correlation Coefficients (ICC) of the Inverse Simpson Index of twin pairs versus all 22 individuals. (C) Histogram of inverse Simpson indices for the total NK cell population of each donor. (D) Total phenotypes predicted by the Chao 1 non-parametric species estimator (calculated as described in Methods) for all donors.

Figure 7. Calculating the expanse of the NK cell repertoire

(A) Sample-based rarefaction predicting the total number of NK cell phenotypes within the cohort of 22 donors. The number of phenotypes observed was calculated for incremental inputs of 50,000 NK cells up to and including the total 304,909 NK cells sampled in all 22 donors, reshuffling the order of donor sampling each of 10 times. These data were used to plot a curve, which when extrapolated to an asymptote provided an estimate of the total NK cell population. (B) Non-parametric species estimators confirm quantification of the total number of NK cell phenotypes.