Coordinated Regulation of NK Expression in the Maturing Human

This information is current as Dara M. Strauss-Albee, Amir Horowitz, Peter Parham and of October 2, 2021. Catherine A. Blish J Immunol 2014; 193:4871-4879; Prepublished online 6 October 2014; doi: 10.4049/jimmunol.1401821 http://www.jimmunol.org/content/193/10/4871 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2014/10/03/jimmunol.140182 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 46 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/193/10/4871.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Coordinated Regulation of NK Receptor Expression in the Maturing Human Immune System

Dara M. Strauss-Albee,*,† Amir Horowitz,*,‡,x Peter Parham,*,‡,x and Catherine A. Blish*,†

NK cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non–Ag-specific features termed “altered self” through combinatorial signals from activating and inhibitory receptors. These NKRs are also expressed on CD4+ and CD8+ T cells, B cells, and monocytes, although a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKRs are more likely to be expressed on non-NK cells, especially CD8+ T cells. Mature NK and CD8+ T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, whereas CD8+ T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor leuko- Downloaded from cyte Ig–like receptor subfamily b member 1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of “altered self” recognition potential among leukocyte lineages. This likely reduces Ag specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection. The Journal of Immunology, 2014, 193: 4871–4879. http://www.jimmunol.org/ atural killer cells quickly recognize and kill cells that Diverse surface marker expression is not unique to NK cells. display features of “altered self,” including cells that After activation, proliferation, differentiation, and subsequent N are virus infected, malignant, or stressed. NK cells are maturation, many leukocytes develop into distinct subsets whose stimulated through a paradigm markedly different from that of diversity correlates with vast functional heterogeneity (1). In other lymphocytes. A balance of combinatorial signaling inputs addition to markers specific to each lineage, NKRs are also mediated through a collection of activating and inhibitory recep- expressed on other cell types found in human peripheral blood, tors, collectively termed NKRs, determines NK cell activation including T cells, B cells, and monocytes (2–5). This phenotypic status. These receptors include the killer Ig–like receptors (KIRs) correspondence mediates functional resemblance (6–8). by guest on October 2, 2021 that recognize HLA-A, -B, -C, and other ligands, leukocyte Ig– Like NKRs, some markers of maturation can also be compared like receptor subfamily b member 1 (LILRB1) that binds a con- across populations because of their broad expression patterns. served region in all HLA class I , natural cytotoxicity A relevant example is CD57, a terminally sulfated carbohydrate receptors that recognize a variety of pathogen, tumor, and self- epitope that irreversibly marks maturity in both NK cells (9) and derived ligands, C-type-lectin–like receptors that recognize CD8+ T cells (10). Functionally, CD57+ NK and CD8+ T cells are HLA-E, and signaling lymphocyte activation molecule family less proliferative, less responsive to cytokine stimulation, and have receptors that recognize a variety of ligands and are critical for a higher cytolytic capacity than CD572 cells (11, 12). In young immune regulation. and middle-aged individuals, CD57+ CD8+ T cells may also be more polyfunctional than CD572 cells (13). CD57 also has medical relevance, as its acquisition has been documented both in *Stanford Immunology, Stanford University School of Medicine, Stanford, CA the context of aging (14, 15) and in chronic infection (reviewed 94305; †Department of Medicine, Stanford University School of Medicine, Stanford, in Ref. 16). CA 94305; ‡Department of Microbiology and Immunology, Stanford University + x CD8 T cells that acquire CD57 also lose expression of CD28 School of Medicine, Stanford, CA 94305; and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305 and acquire expression of inhibitory NKRs, especially KIR and + ORCID: 0000-0003-1235-7800 (D.M.S.-A.). CD94/NKG2A (10, 17, 18). Mature NKR-expressing CD8 T cell Received for publication July 17, 2014. Accepted for publication August 29, 2014. populations are oligoclonal or monoclonal (10, 19), and mRNA expression of NKRs has been shown to be highly heterogeneous This work was supported by National Science Foundation Graduate Research Fellowship DGE-114740 (to D.M.S.-A.), National Institutes of Health (NIH) Training Grant T32 even in cells with identical TCR clonotypes (20). Functionally, AI07290 (to A.H.), NIH Grant AI22039 (to P.P.), a Beckman Young Investigator award NKRs acquired on CD8+ T cells have so far been shown to (to C.A.B.), and NIH Director’s New Innovator Award 1DP2AI112193 (to C.A.B.). function primarily through inhibitory signaling, but activating Address correspondence and reprint requests to Dr. Catherine A. Blish, Division of receptor expression patterns have never been evaluated. Because Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Lane Building, L-134, Stanford, of this ability of NKRs to modify downstream activation status, CA 94305-5107. E-mail address: [email protected] their expression across cell types has significant implications for The online version of this article contains supplemental material. the regulation of immunity. Abbreviations used in this article: KIR, killer Ig–like receptor; LILRB1, leukocyte The determinants that control human NKR expression Ig–like receptor subfamily b member 1; NIH, National Institutes of Health; PCA, across immune cell types have not been examined. To fully un- principal component analysis. derstand the functional significance of NKR acquisition on CD8+ Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 T and other cells, it is necessary to concurrently evaluate the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401821 4872 MATURITY-DRIVEN REGULATION OF HUMAN NKR EXPRESSION combinatorial protein expression of all types of NKRs at single- monozygotic twins deviate significantly in their total NK cell cell resolution. No prior studies have simultaneously evaluated repertoire (24), we considered each pair as separate individuals for both activating and inhibitory NKR expression patterns across the purposes of this analysis. leukocyte lineages. Human studies are also essential to recognize the potential implications of these receptor expression patterns in Fidelity of NKR for NK cells vaccine and therapeutic strategies, especially given the vast phe- We designed a gating scheme to distinguish five major leukocyte notypic differences between murine and human NKR systems. types present in human peripheral blood: CD4+ and CD8+ T cells, The recently developed high-dimensional analysis platform B cells, monocytes, and NK cells (Supplemental Fig. 1). To de- known as mass cytometry allows for single-cell evaluation of termine phenotypic marker distribution across populations, we 40+ parameters. Since its inception, this technology has vastly ex- calculated the frequencies of surface markers on each cell type for panded our understanding of the human immune system (21–23). It each of the 22 individuals. This shows that several receptors, such has also deepened our comprehension of the astonishing diversity as NKp46 and NKG2A, are expressed almost exclusively in NK of the human NK cell repertoire (24). In addition to analyzing cells, whereas others, notably LILRB1, are expressed at a range of NKRs on NK cells, mass cytometry also permits their examination levels across all five cell types (Fig. 1A). across multiple cell types in the controlled environment of a single We next sought to delineate which receptors were expressed stained sample. In this study, we used this technology to analyze more highly on NK cells than on the other cells. We gave each the expression of activating and inhibitory NKRs on NK cells, receptor a score of its “fidelity” for NK cells by dividing its fre- T cells, B cells, and monocytes in peripheral blood from 22 quency of expression on NK cells by its frequency of expression healthy donors. We sought to determine how the expression of on all cells. Taking into account data for each receptor for each NKR on NK cells and other cell types might reflect the importance donor, we ranked receptors from least to most median NK fidelity Downloaded from of “altered self” detection in diverse branches of the maturing (Fig. 1B). Although CD56 is the most commonly used NK cell human immune system. marker, we found that NKp46 (Fig. 1B, far right) is the most NK cell–specific receptor, confirming and extending previous results Materials and Methods (25). This analysis also identified a set of 17 receptors that are Human subjects and cells more frequently expressed on NK cells than on the total cell population and can be used to define NK cells (Fig. 1B, red shaded http://www.jimmunol.org/ This study was performed on cryopreserved human PBMCs in accordance box). This set of receptors includes the KIRs, natural cytotoxicity with the Declaration of Helsinki and was approved by the Institutional Review Boards of Stanford University and SRI International. For details of receptors, and other canonical NKRs such as CD122, CD16, and sample sourcing, see Horowitz et al. (24). In brief, subjects included 10 NKG2D, but does not include LILRB1, because of its high fre- healthy unrelated adults (5 male and 5 female adults, aged 24–60 y, median quency of expression by monocytes and B cells. age 34 y), 2 anonymous blood bank control subjects, and 5 sets of We identified considerable variability among donors in the monozygotic twins (4 male and 6 female twins, aged 20–40 y, median age fidelity of NKRs for NK cells, as is indicated by the broad 23 y). Nine of 21 (42.9%) subjects tested were CMV seropositive. All subjects gave fully informed and written consent. interquartile ranges in the boxplot in Fig. 1B. For some receptors, such as KIR2DS4, surface expression is highly genotype driven. by guest on October 2, 2021 Staining and data acquisition For example, donors with a functional, surface-expressible allele PBMCs were thawed and washed with RPMI 1640 (Corning Cellgro) with of the KIR2DS4 show high fidelity of KIR2DS4 for NK cells 10% heat-inactivated FBS, 2 mM L-glutamine, and antibiotics (100 U/ml (Fig. 1B, green box), but donors without a functional KIR2DS4 penicillin, 100 mg/ml streptomycin; Life Technologies BRL/Life Tech- gene do not show KIR2DS4 fidelity for NK cells, because they do nologies) and rested at 37˚C with 5% CO2 for 4 h. Two million PBMCs not express the protein (Fig. 1B, blue box) (24). However, vari- were stained for mass cytometry analyses as described previously (21, 22, 24). Data were acquired on a CyTOF instrument (DVS Sciences). Data ability in fidelity was observed even among receptors with little files of mass cytometry analysis of PBMCs in .fcs format for each indi- genetic polymorphism, such as NKG2A, NKG2C, and NKp46, vidual are available for download in ImmPort (https://immport.niaid.nih. suggesting that genotype alone is not sufficient to account for the gov/) under access number SDY232. differences. Analytics Cellular population distribution of NK cell markers FCS files were analyzed using FlowJo software (Tree Star). Boolean To understand how the NKRs cluster among different cellular analyses were performed using a custom Python script (http://www.python. org) to sort cells based upon positive or negative expression of each subsets, we created a correlation matrix of 21 lineage markers and marker. The inverse Simpson index was calculated using the following NKRs based on concatenated data from all detected live cells from equation: all 22 individuals (Fig. 2A). Hierarchical clustering of these re- 1 ceptor coexpression patterns resulted in several distinct clusters D ¼ + S consistent with defined cellular lineages, including CD4 T cells, 2 + + pi B cells, and monocytes. The CD4 T cell cluster is enriched for i¼ 1 expression of CD3, CD4, CD127, CD27, and CCR7. CD19, the where S is the total number of species and pi is the proportional abundance only B cell marker included in the panel, is not highly coexpressed of the ith species. All statistical analyses were performed using the open with any other markers except HLA-DR. Cells coexpressing source statistical package R (http://www.r-project.org). CD33, HLA-DR, LILRB1, and 2B4 form a distinct monocyte-like cluster. The LILRB1 and 2B4 receptors are also highly expressed Results on subpopulations of NK cells. The remaining 11 receptors also We previously used a mass cytometry platform of 37 lineage form a distinct cluster encompassing many NKRs that are highly markers and NKRs to interrogate the human NK cell repertoire expressed on NK cells. This cluster also has two distinct sub- (24). In this study, we used this approach to examine the ex- groups, formed by canonical NK cell markers (CD94, CD122, pression of these markers on 5 leukocyte populations in the pe- NKp30, CD56, CD16, NKp46, and NKG2A) and mature NK cell ripheral blood from a cohort of 12 healthy unrelated individuals markers (CD57, NKG2C, and NKG2D). Unexpectedly, CD8 also and 5 sets of monozygotic twins. Because we have shown that clusters with the mature NK markers, rather than with the CD4+ The Journal of Immunology 4873 Downloaded from

FIGURE 1. Restricted cell-type expression patterns of NKRs and lineage markers. (A) Frequencies of NKRs on each cell type (designated by unique colors and symbols), as determined by Boolean gating, for each of 22 donors. http://www.jimmunol.org/ (B) Fidelity of each receptor for NK cells, as measured by log(frequency on NK cells/frequency on all live cells). KIR2DS4+ and KIR2DS42 individuals (as determined by genotyping) are annotated in green and blue boxes, re- spectively. Receptors with a log fidelity score .0 were called as NK-specific receptors (red shaded box). by guest on October 2, 2021

T cell cluster, even though it is also highly coexpressed with CD3, extreme diversity of NKR expression. Despite this complexity, CD27, and CD127. This analysis therefore shows that, when the analysis identified three informative components that together assessed by NKR expression, CD8+ T cells are more similar to NK account for 40.8% of the variance in NKR expression patterns cells than to CD4+ T cells. (Fig. 2C). NK cell markers, including CD56, NKp46, CD94, To understand better the expression of NKRs by the divergent NKG2A, CD16, and CD122, contribute most significantly to lineages of leukocyte, we performed a principal component component 1, whereas components 2 and 3 are mainly shaped by analysis (PCA) to reduce the dataset to a small number of com- the contributions from monocyte and T cell markers (Fig. 2B, 2C). ponents that can explain much of the variation (Fig. 2B). In these Like the hierarchical clustering, the PCA cleanly separated cell data, variance is spread among many components because of the populations, but additionally provided insight to the positions of 4874 MATURITY-DRIVEN REGULATION OF HUMAN NKR EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 2. Cell population distributions distinguish roles of NKRs and lineage markers. (A) Hierarchical clustering of a correlation matrix of receptor expression patterns. The Spearman’s correlation, representing the likelihood that each pair of markers is coexpressed on the same cell, is shown on a scale of 21 (red) to 1 (blue). This matrix is built on data from all 22 individuals. (B) Three-dimensional projection of the first three components of a PCA of receptors from all individuals, annotated by cell type. (C) Summary of the contributions of each marker to each of the first three components of the PCA displayed in (B). The size and color of the circle represent that marker’s contribution to each of the first three components on a scale of 20.4 (red) to 0.4 (blue). receptors that did not fit into cell-type categories. For example, of the cohort-wide top 17 most NK cell–specific receptors. We NKG2D segregates between canonical NK cell markers and CD8, used the level of CD57 expression on all leukocytes (although it is indicative of its expression on both cell types (Fig. 2B). Further, primarily expressed on NK and CD8+ T cells) as a measure of the this analysis revealed a T cell–NK cell spectrum consisting of maturity of each donor’s total cellular repertoire, because this CD4+ T cells, CD8+ T cells, mature NK cells, and canonical NK molecule is known to accumulate in settings of immune maturity cells. These data help to explain the unexpected position of CD8 including age and immune activation (27). We found that this in Fig. 2A by showing that CD8+ T cells and mature NK cells are trend is broadly generalizable: in our cohort of 22 donors, the intermediates on a continuum with CD4+ T cells and canonical maturity of the cellular repertoire, as measured by the frequency NK cells at either end (Fig. 2B). of CD57 expression on all live cells, is inversely correlated with the NK cell fidelity score (Spearman’s s = 20.70, p = 4.2 3 1024; Determinants of NKR fidelity Fig. 3B). The NK cell fidelity score also closely correlates with To visualize better the distribution of NKR on each cell type, we the frequency of the costimulatory receptor CD27, which is pro- plotted the average frequencies of each NKR and lineage marker on gressively lost during differentiation of T and NK cells (Spear- each cell type in a series of petal plots (Fig. 3A). In this study, we man’s s = 0.61, p = 0.0031; Fig. 3C). discovered a unique CD8+ T cell expression signature with re- To determine the degree to which the fidelity–maturity link semblance to both NK cells and CD4+ T cells. We also observed is driven by maturing NK cells expressing fewer NKRs and that LILRB1 is the primary NKR expressed on monocytes and maturing non-NK cells acquiring NKR, we used Boolean B cells. analysis to categorize the expression of each receptor on each Based on previous reports on KIR acquisition by memory CD8+ cell as positive or negative, allowing quantification of the total T cells (10, 20, 26), we hypothesized that NKR expression on non- number of receptors on a per-cell basis. We used these data to NK cells was related to the state of cellular differentiation. We compare the maturity-driven gains in total number of receptors therefore calculated an NK fidelity score for each donor by av- on NK cells and also on CD8+ T cells, which express the most eraging the NK cell fidelity (the value plotted in Fig. 1B) of each NKRs of all non-NK cell types (Fig. 3A). For both CD8+ T The Journal of Immunology 4875 Downloaded from http://www.jimmunol.org/

FIGURE 3. NK cell specificity of the marker repertoire is inversely correlated with immune maturity. (A) Petal plots representing the average frequency (among 22 individuals) of each receptor’s expression on each cell type. Receptors are filled according to their class (blue = activating/lineage, red = inhibitory, violet = other lineage). Scale is from 0 (center of plot) to 100% (outer edge of plot) receptor expression frequency on the indicated cell type. (B and C) Plots of frequencies on all live cells of CD57 and CD27, respectively, versus NK cell fidelity score, as calculated by the mean fidelity of the NK- specific receptors from Fig. 1B, for each of 22 individuals, with Spearman’s correlations displayed. (D) The total number of NKRs measured per cell (as determined by Boolean gating) on CD572 and CD57+ CD8+ T and NK cells. Paired measurements of NKRs per cell are shown for each of 22 individuals, with Wilcoxon signed- statistics. by guest on October 2, 2021

(Wilcoxon signed-rank p =4.83 1027) and NK cells (Wil- To understand how NKR expression patterns and the individual coxon signed-rank p =3.63 1024), more receptors are cellular combinations of receptors differed among cellular lineages, expressedonCD57+ cells than on CD572 cells (Fig. 3D). we used a well-established method borrowed from ecology to Maturing CD8+ T cells gain an average of 1.99 receptors per quantify diversity. We used Boolean analysis to classify a cellular cell, compared with only 0.59 receptors for NK cells. Despite “species” based on the expression of a unique combination of its ability to skew the immune repertoire, donor serostatus for receptors. We then considered each cell type to be a “population.” CMV is not sufficient to explain the acquisition of NKRs on This allowed us to quantify the diversity of NKRs on each cell type either cell population: CMV+ and CMV2 donors are indis- using the inverse Simpson Index, which accounts for both the dis- tinguishable in the extent to which their mature CD8+ Tand tribution and the total number of species in a population (29). We NK cells upregulate NKR (Supplemental Fig. 2). Thus, as the find that NK diversity correlates with repertoire maturity (Spear- immune response becomes more mature, CD8+ T cells broaden man’s s = 0.44, p = 0.043; Fig. 4B). As predicted by their acqui- their repertoire by acquiring NK cell machinery. This is also sition of a substantial number of NKRs in Fig. 3D, CD8+ T cells also true when CD8+ T cell maturity is measured by loss of CCR7 show a strong significant correlation of repertoire maturity with di- and CD27 (28) (Supplemental Fig. 3). versity (Spearman’s s =0.69,p =5.33 1024). Monocytes, B cells, and CD4+ T cells show no correlations of maturity with NKR di- Differential NKR regulation versity. Therefore, although they have distinct patterns, both CD8+ T + Because maturing CD8 T cells acquire NKRs and become and NK cells diversify their NKRs as the cellular repertoire matures. intermediates on the NK cell–T cell continuum, as shown in Fig. 2, we next examined whether single-cell patterns of pheno- Determinants of phenotypic divergence of NKRs across typic expression of NKRs could distinguish CD8+ T and NK cells. immune populations The Boolean data divide the population of cells into discrete To understand the differential diversification of NK and CD8+ subpopulations, so we used a multivariate dimensionality reduc- T cells during maturity, we divided both the NK cell and the CD8 tion tool known as correspondence analysis, which is designed for T cell populations into two subpopulations, according to their ex- such noncontinuous incidence data. Performing this analysis on pression of the cellular maturity marker CD57. We then compared each of the five cell types from each donor revealed two primary the difference in frequency of each receptor between CD572 and components that readily separated CD8+ T and NK cells (Fig. 4A). CD57+ cells (Fig. 5A). Compared with immature CD572 NK cells, CD8+ T cells therefore express unique combinations of NKRs, mature CD57+ NK cells (Fig. 5A, filled sections) express higher forming a distinct, non-NK cell niche. levels of KIR2DL1, KIR2DL3, NKG2C, and CD16, LILRB1, 4876 MATURITY-DRIVEN REGULATION OF HUMAN NKR EXPRESSION

To understand how these changes are mediated on a single-cell basis, we calculated the number of inhibitory receptors on each of these four populations of CD572 and CD57+ CD8+ T and NK cells (Fig. 5B). Both cell types are similar in their acquisition of in- hibitory receptors with maturation: maturing CD8+ T cells gain a mean of 0.55 inhibitory receptors per cell, whereas NK cells gain 0.31 (summarized in Fig. 5D). Notably, the distribution of inhibitory receptors on mature CD57+ CD8+ T cells and immature CD572 NK cells is markedly similar. Both NK and CD8+ T cells gain activating receptors with maturity, but the difference in magnitude is striking (Fig. 5C, 5D). Maturing CD8+ T cells gain a mean of 1.43 activating receptors per cell, whereas NK cells gain just 0.28. Thus, CD8+ T cells gain both activating and inhibitory potential when they mature, whereas NK cells acquire primarily inhibitory potential. Finally, we examined maturity-driven changes in LILRB1, which was unique among receptors examined in its high level of expression on multiple leukocyte types. This receptor is expressed on monocytes and B cells (Fig. 1A), and is highly upregulated on maturing CD8+ T cells (Fig. 5A). We therefore asked whether Downloaded from LILRB1 expression on monocytes and B cells relates to NK and CD8+ T cell repertoire maturity. With increasing CD57 levels, LILRB1 is significantly increased on CD8+ T cells (Spearman’s s = 0.69, p = 3.4 3 1024; Fig. 5E). Remarkably, it is concurrently significantly decreased on monocytes (Spearman’s s = 20.46, p = 0.032) and B cells (Spearman’s s = 20.64, p = 0.0012; Fig. 5E). http://www.jimmunol.org/ Taken together, our observations of simultaneous acquisition and loss of NKRs across multiple cell types suggest a coordinated regulation of NKR expression in the maturing human immune system.

Discussion We used mass cytometry to identify the selective expression of

activating and inhibitory NKRs in human PBMCs. We found that by guest on October 2, 2021 NK, monocyte, CD4+ T, CD8+ T, and B cell populations form distinct populations based on their surface NKR expression pat- terns. Our data reveal that, as the immune repertoire matures, NKRs are more likely to be found on non-NK cells, especially CD8+ T cells. Our findings further demonstrate that the diversity of the NKR repertoire on both CD8+ T and NK cells, but not other leukocytes, increases with maturity. However, based on their FIGURE 4. Determinants of diversity in NK cells and CD8+ T cells. (A) single-cell NKR combinatorial expression patterns, we show for Correspondence analysis of all Boolean subpopulations from all cell types the first time, to our knowledge, that these two cell types are (designated by unique colors and symbols) from all 22 individuals. The driven by differential maturation programs. NK maturation is two mostly highly informative components are shown on the axes. (B) mediated primarily by the acquisition of specific inhibitory Plots of the frequency of CD57+ cells (as a marker of maturity) versus the receptors, especially KIR, whereas CD8+ T cells acquire both diversity of NKRs on each cell type, as measured by the Inverse Simpson activating and inhibitory receptors, especially those used for Index. Significant associations by Spearman’s correlation are annotated. generalized recognition. Finally, we show that monocyte and B cell populations downregulate LILRB1 as CD57 accumulates in KIR3DL1, KIR2DS4, 2B4, and NKp30. They also express lower the total cellular repertoire. These data indicate that the expression levels of NKG2A, NKG2D, NKp46, and CD122. These gains and of NKR is coordinately regulated on divergent cell types as the losses are not dominated by any single receptor or set of receptors. human immune system matures. By contrast, compared with immature CD8+ T cells, mature CD8+ The acquisition of both activating and inhibitory receptors on T cells (Fig. 5A, unfilled sections) show increased expression of CD8+ T cells as they mature suggests increasing reminiscence of six primary receptors: LILRB1, 2B4, NKp30, CD56, CD94, and NK cells. Although their inhibitory receptor acquisition does in- CD122. Mature CD8+ T cells also express slightly higher levels of clude specific inhibitory KIRs, it is mediated mainly by upregu- all remaining NKRs and, unlike NK cells, do not show reduced lation of generalized recognition receptors, especially LILRB1, expression of any NKRs. This analysis therefore revealed matu- NKG2A, and 2B4. Increased inhibitory capacity could help to ration programs unique to each cell type. The changes in fre- explain recent functional data showing that, contrary to long-held quency associated with the gain of CD57 on NK cells were similar dogma, memory CD8+ T cells actually require a higher Ag con- to those found on the recently described “memory-like” NKG2C+ centration for activation than do naive CD8+ T cells (30). It has NK cells, with notable differences including increased NKp30 and also been suggested that the presence of inhibitory receptors decreased NKG2D on CD57+ NK cells (Supplemental Fig. 4). renders CD8+ T cells less susceptible to activation-induced cell The Journal of Immunology 4877 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Differential maturation programs of NK and CD8+ T cells. (A) The difference of each receptor’s frequency on CD57+ versus CD572 cells, averaged across 22 individuals. Filled bars represent NK cells and unfilled bars represent CD8+ T cells, with inhibitory receptors in red and activating receptors/lineage markers in blue for both cell types. Scale represents the change in the frequency of expression on CD57+ versus CD572 cells from 210% (10% less expression on CD57+ cells) to 30% (30% more expression on CD57+ cells). (B and C) Total number of inhibitory receptors (B) or activating receptors/lineage markers (C) on CD572 and CD57+ CD8+ T and NK cells. (D) Summary box plot showing the per-cell count of each receptor type on each cell population. (E) Spearman’s correlation of CD57 frequency on all live cells and LILRB1 frequency on each cell type. death, allowing them to be selectively maintained (31, 32). B7-H6, which is expressed on tumor cells (35), and BAG3, which Dampening the activation potential of each individual clone could is released from tumor cells (36) and immature dendritic cells also prevent aberrant reactivity and promote response optimiza- (37). On a single-cell level, as CD8+ T cells become more re- tion by non–Ag-specific signals in a differentiated population of fractive to stimulation through the TCR, perhaps it is advanta- CD8+ T cells that is known to be both oligoclonal (19) and cross- geous for them to acquire such additional non–Ag-specific roles in reactive (33). Further, the acquisition of noncanonical, NK-like the immune response by acquiring the machinery of NK cells. roles of CD8+ T cells is not without precedent, because these Although CD8+ T cells become more NK cell–like as they cells have recently been shown to contribute to control of infec- mature, our data demonstrate that their specific maturity program tions and tumors in an HLA-E–restricted manner (34). diverges from that of NK cells. This finding is consistent with Along with inhibitory receptors, maturing CD8+ T cells also literature showing distinct transcriptional regulation of KIR (38) acquire activating receptors, and the increase in NKp30 is espe- and differing patterns of surface KIR expression (10) in these cell cially striking. In addition to its roles in viral recognition, NKp30 types. It is also logical based on the context of cellular activa- has been shown to specifically activate by binding to its ligands tion triggers. CD8+ T cells require Ag-specific stimulation whose 4878 MATURITY-DRIVEN REGULATION OF HUMAN NKR EXPRESSION cross-reactivity may need to be checked during immune aging. these cell types is influenced by multiple factors, which may in- NK cells do not require Ag-specific stimulation; therefore, creat- clude other viral infections or environmental stimuli. Thus, the ing new combinations of receptors may be sufficient for recognition pathogen-rich environment of human existence resembles a state of a diverse assortment of Ags. In addition, NK cells continue to of induced chronic infection in mice. This has important impli- acquire inhibitory receptors while showing little change in the cations for the interpretation of murine immunology studies. number of activating receptors, which could help to avoid aberrant Chronic infection models in mice may actually be more relevant reactivity during their maturation. to the human condition than the murine steady-state. The increased diversity of NKR expression on NK and T cells We therefore propose that a maturing immune system becomes during maturation may be an adaptive mechanism to maintain increasingly Ag independent. CD8+ T cells require higher con- a robust immune response. A decrease in T cell AgR diversity centrations of Ag to overcome inhibitory signals and achieve during immune maturation has been well-documented (39–42) and stimulation, and may additionally acquire non–Ag-specific roles may contribute to age-related immunodeficiency (43) and re- through upregulation of activating NKRs. Concurrently, maturing sponse to vaccination (44). Thus, the increased diversity of NKR NK cells diversify receptor combinations present in their reper- on both NK and CD8+ T cells with maturity suggests that NKR toire, enhancing their surveillance potential, whereas monocytes, expression is a uniquely regulated process that maintains a diverse the precursors to DCs, have a lower activation threshold and may pool of responders. NK and cytotoxic CD8+ T cells have similar be less able to present Ag. This shift may have evolved when effector functions, which are performed principally by NK cells in humans lived in isolated communities, where the probability of the innate immune response and by CD8+ T cells in the adaptive encountering novel Ags decreased with increasing age, shifting the immune response. As the adaptive immune response becomes priority of the mature immune system to maintaining homeostasis. more mature, the broadening and increasing sophistication of the In the modern era, where transportation creates a constant on- Downloaded from CD8+ T cell response could potentially compensate for the de- slaught of novel Ags, this property may help explain the increased creasing diversity in AgRs. immunodeficiency observed in human aging. These dramatic CD57 accumulates on NK cells (14) and CD8+ T cells (45) as effects of maturity on the human immune NKR repertoire should humans age, and has been documented in a variety of infections. be considered in the design and administration of vaccines and Because our analyses addressed the changes in NKR expression therapeutics, as Ag-specific responses may require boosting both occurring with CD57 acquisition, our findings have implications innate and adaptive immune responses in an aging population. http://www.jimmunol.org/ for understanding the immune system in a state of both aging and chronic infection. Because monocytes and B cells express little to Acknowledgments no CD57 (Fig. 1), it has been difficult to discern how changes We thank Drs. Natalie Bezman, Mark Davis, and Holden Maecker for in their receptor expression correlate with this maturity marker. helpful discussions and comments on the manuscript. In this study, we took advantage of CD57 expression on NK and T cells, measured simultaneously through mass cytometry, to understand the expression of NKRs on these cells as the total Disclosures immune repertoire matures. We find that monocytes and B cells The authors have no financial conflicts of interest. by guest on October 2, 2021 show progressively less LILRB1 expression in more mature rep- ertoires, whereas CD8+ T cells acquire more. LILRB1 expression References has been associated with Ag presentation capability in monocyte- 1. Mahnke, Y. D., T. M. Brodie, F. Sallusto, M. Roederer, and E. Lugli. 2013. The derived dendritic cells (46), so it is possible that this function is who’s who of T-cell differentiation: human memory T-cell subsets. Eur. J. Immunol. 43: 2797–2809. decreased as the repertoire matures. Lowering the threshold for 2. Lamar, D. L., C. M. Weyand, and J. J. Goronzy. 2010. 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