Novel Insights on Human NK Cells' Immunological Modalities Revealed by Gene Expression Profiling

This information is current as Jacob Hanna, Pamela Bechtel, Yufeng Zhai, Fadi Youssef, of September 24, 2021. Karen McLachlan and Ofer Mandelboim J Immunol 2004; 173:6547-6563; ; doi: 10.4049/jimmunol.173.11.6547 http://www.jimmunol.org/content/173/11/6547 Downloaded from

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Novel Insights on Human NK Cells’ Immunological Modalities Revealed by Gene Expression Proﬁling1

Jacob Hanna,* Pamela Bechtel,† Yufeng Zhai,† Fadi Youssef,* Karen McLachlan,† and Ofer Mandelboim2*

As part of the innate immune system, human NK cells play a critical role early in the systemic host defense against pathogens and tumor cells. Recent studies suggest a more complex view of NK cell behavior, as different functions and tissue localizing capabilities seem to be preferentially assigned to distinct subpopulations of NK cells, CD56dimCD16؉ or CD56brightCD16؊. In this study, we used oligonucleotide microarrays to compare the expression profile of ϳ20,000 genes in three NK cell subpopulations: peripheral blood-derived CD56dimCD16؉, CD56brightCD16؊, and in vitro-activated CD16؉ NK cells. The differential expression of selected genes was verified by flow cytometry and functional assays. When comparing CD56dimCD16؉ and CD56brightCD16؊ subsets, a new heterogeneous molecular basis for the functional and developmental differences between these two subsets was revealed. Downloaded from Furthermore, systematic analysis of transcriptional changes in activated CD16؉ NK cells provided us with a better understanding of NK function in inflamed tissues. We highlight a number of genes that were overexpressed upon activation (e.g., OX40 ligand, CD86, Tim3, galectins, etc.), that enable these cells to directly cross-talk with other innate and adaptive immune effectors. The overexpressed genes assign novel intriguing immunomodulatory functions to activated NK cells, in addition to their potent cytotoxic abilities. The Journal of Immunology, 2004, 173: 6547–6563. http://www.jimmunol.org/ atural killer cells are a major component of the innate important players in regulating and priming immune responses via immune system having the ability to kill infected target cytokine-mediated cross-talk with neighboring dendritic (DC) and tumor cells and, in addition, to secrete various ef- and T cells (6, 10). In contrast, the more cytotoxic subset, N dim ϩ fector molecules (1, 2). The physiological functions of NK cells CD56 CD16 , has higher levels of CXCR1 and CX3CR1 che- are tightly regulated by a delicate balance of signals transmitted by mokine receptors (7, 8), and therefore is preferentially recruited to activating and inhibitory receptors (3). Human peripheral blood sites of inflammation (4). Signals transduced by locally secreted NK (pbNK)3 cells can be divided into two subsets based on their inflammatory cytokines and specific encounters with target cells cell surface density of CD56 (N-CAM) and CD16 (Fc␥RIII). The synergize to induce activation of this subset. Such activation in- by guest on September 24, 2021 majority of pbNK cells (Ͼ90%) are phenotypically characterized creases dramatically the cytotoxic ability of these cells mainly by dim ϩ bright as CD56 CD16 , while the remaining cells are CD56 up-regulation of natural cytotoxicity receptors (NCRs), costimula- Ϫ CD16 (4). The fact that each of these subsets expresses a unique tory molecules, and granzyme expression (1, 2, 11). repertoire of chemokine receptors and adhesion molecules encour- The above observations, together with the robust abundance of aged several groups to characterize the NK cell pool in secondary CD56brightCD16Ϫ NK cells in secondary lymphoid organs, have lymphoid tissues and inflammation sites (5–7). prompted scientists to try to understand and characterize differ- bright Ϫ Indeed, it was found that the CD56 CD16 NK cell popu- ences between the different NK subsets. In addition, the early relation preferentially expressed CD62L and chemokine receptors cruitment and activation of cytotoxic NK cells in inflammatory CCR7, CXCR4 (6–8). This might explain why this subset is en- sites raises many questions regarding potential novel immune riched in various human secondary lymphoid organs (lymph functions of these cells and unique cross-talk pathways between nodes, tonsils, and spleen) (5, 6). Interestingly, this subset has innate and adaptive immune responses used by this subset that relatively lower cytotoxic abilities but enhanced cytokine respon- extend beyond their cytotoxic capabilities. siveness and IFN-␥ secretion capabilities (6, 9). In correlation with bright Ϫ In the present study, global patterns of gene expression in these observations, CD56 CD16 cells have been shown as Ϫ ϩ freshly isolated CD56brightCD16 , CD56dimCD16 , and in vitro- activated CD16ϩ NK cells were compared by using oligonucleo- *The Lautenberg Center for General and Tumor Immunology, Hebrew University- tide microarrays. The transcriptional profiles provide detailed Hadassah Medical School, Jerusalem, Israel; and †Biogen Idec, San Diego, CA 92191 information on the gene expression differences between CD56bright Ϫ dim ϩ Received for publication June 28, 2004. Accepted for publication August 12, 2004. CD16 and CD56 CD16 cells and promote better understand- The costs of publication of this article were defrayed in part by the payment of page ing of the biological behavior for each of these subsets. Addition- charges. This article must therefore be hereby marked advertisement in accordance ally, we studied the gene expression changes in CD16ϩ NK cells with 18 U.S.C. Section 1734 solely to indicate this fact. following in vitro activation. We describe the induction of several 1 This research was supported by research grants from the Israel Cancer Research Foundation, the Israel Science Foundation, the European Commission (QLK2-CT- novel membrane-bound and secreted immune effector molecules 2002-011112), the U.S.-Israel Binational Science Foundation, and the Fritz Thyssen that are important in orchestrating the innate and adaptive immune Foundation. responses. We have also selected some of the differentially ex- 2 Address correspondence and reprint requests to Dr. Ofer Mandelboim, The Laut- pressed proteins and validated their expression by flow cytometry enberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. E-mail address: [email protected] and functional assays. Finally, we discuss the possible impact of 3 Abbreviations used in this paper: pbNK, peripheral blood NK; DC, dendritic cell; NCR, several observed transcription patterns in NK subsets on fundamental natural cytotoxicity receptor; EAE, experimental autoimmune encephalomyelitis. immunological scenarios like infection and immune surveillance.

Materials and Methods When examining the reproducibility of normalized duplicates for each 2 Ͼ Preparation of NK subset samples of the three NK samples, there was a significant correlation (R 0.98) (Fig. 1, F–H). Additionally, examination of genes up- or down-regulated PBLs were isolated from different healthy donors using Ficoll gradients. by at least 2-fold between any two normalized data sets from different NK Isolation of NK cells was performed using the NK Isolation kit II (Miltenyi samples always showed a statistically significant correlation in 12 possible Biotec, Auburn, CA), according to manufacturer’s instructions. A total of combinations examined (R2 Ͼ 0.992, data not shown). This confirmed the 100 ϫ 106 cells enriched for CD56ϩCD3Ϫ NK cells from each donor were reproducibility of the 2-fold change criteria between different NK subsets. subsequently sorted as described below to purify CD56dimCD16ϩCD3Ϫ This reproducibility allowed normalized values for each replicate to be and CD56brightCD16ϪCD3Ϫ subsets (see Fig. 1A). Purified cells were averaged. Genes were classified as up- or down-regulated if they demon- pooled after isolation and mRNA was extracted immediately and stored at strated at least a 2-fold alteration in expression level. Ϫ70°C. Bulk polyclonal in vitro-activated NK cultures were prepared from For Venn diagrams, three paired comparisons were made between NK Ϫ ϩ ϩ CD56dimCD16ϩCD3Ϫ sorted cells from nine donors and were grown on subsets: 1) CD56brightCD16 vs CD56dimCD16 ; 2) activated CD16 NK ϩ ϩ Ϫ feeder cells in medium supplemented with human IL-2 and PHA as pre- vs CD56dimCD16 ; 3) activated CD16 NK vs CD56brightCD16 . Venn viously described (2). Activated NK cells were only used after repurifica- diagrams were constructed by intersecting the set of genes up- or down- tion with the NK Isolation kit II to ensure the elimination of any remnants regulated by 2-fold or greater in each of the three comparisons. Gene func- of irradiated feeder cells (see Fig. 1B). tional classification was based on information available in the Gene On- tology Consortium functional annotations (www.geneontology.org), Flow cytometry and cell sorting Locuslink (www.ncbi.nlm.nih.gov/LocusLink), OMIM (www.ncbi.nlm. nih.gov/entrez/query.fcgi?db-OMIM), and PubMed (www.ncbi.nlm.nih. The following mouse anti-human mAbs, purified or directly conjugated gov./entrez/query.fcgi) databases. with FITC, PE, or CyChrome C, were used in flow cytometric analysis: anti-CD56, anti-CD9 (DAKO, Carpinteria, CA); anti-CD16, anti-CD3, anti- Cytotoxicity assay

CD53, anti-CD81, anti-HLA DR, DP, DQ, anti-CD44, anti-CD62L, anti- Downloaded from galectin 3, anti-CD86, anti-CD70 (all obtained from BD Biosciences, Puriﬁed unactivated NK subsets were sorted as described above. Activated ␣ CD16ϩ and CD16Ϫ NK cells were isolated by the use of anti-CD16 mi- Mountain View, CA); anti-IL-7R -chain (CD127), anti-NKG2D, anti- ϩ Ϫ NKp30, anti-galectin 1 (R&D Systems, Minneapolis, MN); anti-NKp46 crobeads (Miltenyi Biotec) on bulk polyclonal-activated CD56 CD3 NK (12); anti-CD3␨ (cross-reactive to human (13)); anti-OX40L (MBL, cells. The cytotoxic activity of various NK subsets against the 721.221 Nagoya, Japan). For staining and cell sorting, cells were washed in PBS EBV-transformed B cell line was measured after labeling these cells over- 35 supplemented with 2% FCS and incubated with mAb on ice for 30 min, night with [ S]methionine. After labeling, target cells were washed, and 3 followed by washing twice. Cell sorting and ﬂuorescence measurements 5 ϫ 10 labeled target cells were incubated at various E:T ratios. The

35 http://www.jimmunol.org/ were performed on a MoFlo High Performance Cell Sorter (DakoCytoma- killing rate was calculated as percent [ S]methionine release ϭ (cpm sam- tion, Glostrup, Denmark). Data from single cell events were collected using ple Ϫ cpm spontaneous release)/(cpm total Ϫ cpm spontaneous release) ϫ 35 a standard FACSCalibur flow cytometer (BD Immunocytometry Systems, 100. Total [ S]methionine release was measured after incubation of the Mountain View, CA). Data were analyzed using CellQuest (BD Bio- cells with 0.1 M NaOH. In these cytotoxic assays, the spontaneous release sciences). The permeabilization and intracellular staining for galectin1, ga- was Ͻ25% of maximal release. lectin3, and CD3␨ were performed using the Cytofix/Cytoperm Plus kit (BD Pharmingen, San Diego, CA) according to the manufacturer’s T cell costimulation assay instruction. T cell proliferation assays were performed in 96-well flat-bottom plates in triplicate in a final volume of 250 ␮l of RPMI 1640 ϩ 10% human serum. Preparation of labeled RNA and microarray hybridization Plates were coated with a suboptimal concentration of anti-human CD3 Total cellular RNA from the NK subsets pooled from different donors mAb (clone T3D, obtained from the American Type Culture Collection by guest on September 24, 2021 Ϫ ϩ ϩ ␮ (CD56brightCD16 , CD56dimCD16 , and in vitro-activated CD16 NK sub- (Manassas, VA)) (0.1 g/ml in PBS incubated overnight at 4°C). Plates were washed twice with RPMI 1640 before incubating with 25 ϫ 103 sets) was isolated and subjected to a cleanup protocol with RNeasy Mini kits ϩ (Qiagen, Valencia, CA), according to the manufacturer’s specifications. The irradiated (5000 rad) activated CD16 NK or unactivated polyclonal CD56ϩCD3Ϫ NK cells, combined with 50 ϫ 103 autologous CD3ϩ T cells quality of total RNA was assessed using an Agilent Bioanalyzer (see Fig. 1C). ϩ 3 First and second strand cDNA was prepared from a 0.5-␮g RNA template (see (Pan CD3 T Cell Isolation kit; Miltenyi Biotec) for 72 h. [ H]Thymidine (1 ␮Ci/well) was added for the last 24 h of the assays and cells were Fig. 1D), and the cDNA was subjected to in vitro transcription in the presence 3 of biotinylated nucleoside triphosphates. The biotinylated cRNA was frag- harvested to quantitate [ H] incorporation into DNA. Supernatants from mented to uniform sizes (ϳ100 nt as verified in Fig. 1E). CodeLink Uniset proliferation experiments were used for measuring production of human Human 20K I Bioarrays (Amersham Biosciences, Piscataway, NJ) were hy- IL-2 by ELISA using the Quantikine kit (R&D Systems). bridized with each prepared cRNA target in duplicates and stained with Cy5- streptavidin and subsequently washed according to manufacturer’s instruc- Jurkat cell apoptosis assay tions, and then scanned on an Axon GenePix 4000B scanner (Axon A total of 1 ϫ 106 unactivated CD56ϩCD3Ϫ NK cells were grown for 7 Instruments, Foster City, CA). Raw data files were obtained after analysis of days at a concentration of 0.5 ϫ 106/ml in RPMI 1640 medium ϩ 10% heat scanned images with CodeLink expression software (Amersham Biosciences). inactivated FCS with or without the addition of 100 U/ml IL-2 and 1 ng/ml The complete microarray data is deposited at the National Center for Biotech- PHA. Supernatants of these cultures (together with control media contain- nology Information’s (NCBI) Gene Expression Omnibus (GEO) under entry ing the same concentrations of IL-2 and PHA) were added to Jurkat cells names: GSM26200-26205. (1 ϫ 106/ml) for8hat37°C. Induced cell death was measured using the Annexin V Apoptosis kit (Molecular Probes, Eugene, OR) according to Microarray data analysis manufacturer’s instructions. The global normalization (total intensity normalization) method was used for the data obtained from the Amersham array. The mean intensities were Results calculated for each array (Mi, i ϭ 1,2,....,n) and used to calculate the Microarray gene expression analysis of human mean intensity across all arrays (Ma). The scale factors (Fi, i ϭ 1,2, ....,n) CD56brightCD16Ϫ, CD56dimCD16ϩ, and activated CD16ϩ NK were calculated for each array as Ma/Mi. The normalized intensities for each array were calculated by multiplying scale factors by their measured cells intensities. This classical normalization method scales the individual in- The initial aim of this study was to investigate the gene expression tensities so that the mean intensities are the same across all arrays, and profile of ϳ20,000 genes in human CD56dimCD16ϩ NK, were applied to the data by an in-house developed program. Transcripts bright Ϫ ϩ that demonstrated differences Ͼ2-fold in duplicate analysis of the same NK CD56 CD16 , and in vitro-activated CD16 NK cells. These sample examined were excluded from the analysis. The raw intensity data subsets were purified from nine different healthy donors (Fig. 1, A were normalized after exclusion of such genes. Additionally, 20 was set to and B). All samples were re-examined by flow cytometry follow- be the minimal normalized hybridization intensity value (in other words, all ing sorting, to ensure purity above 99% (data not shown). Subse- intensities below 20 were brought up to this value) after observing that a set of more than 10 transcripts (e.g., CD20, CD4, CTLA4, etc.), known to be quently, each purified subset from all donors was pooled and used absent in NK cells, showed hybridization intensity values below this to obtain a total RNA extract. The quality of total RNA used, and threshold. the cRNA that was prepared afterward, was verified by using the The Journal of Immunology 6549 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Global gene expression analysis measured in purified NK subsets. A, Sorting gates applied for purifying NK subsets from pbNK-enriched fractions. After exclusion of CD3ϩ cells, CD56brightCD16Ϫ and CD56dimCD16ϩ subsets were gated. Representative gating conditions on one donor’s cells (of nine performed) is shown. B, Purity of analyzed activated CD16ϩ (CD56ϩCD16ϩ) NK cells was evaluated by flow cytometry staining. High quality of total RNA (C) and cRNA (D) prepared from pooled NK subset samples, together with fragmentation of biotinylated cRNA to uniform size (E), were verified by using an Agilent Bioanalyzer. F–H, Reproducibility of normalized gene expression data was assessed by plotting expression levels from two replicate experiments (A and B) performed on each NK subset. Points falling outside of the lines had expression levels Ͼ2-fold different in one replicate compared with the other. Linear correlation coefficient values (R2) are indicated for each analysis.

Agilent Bioanalyzer (Fig. 1, C and D, respectively). Additionally, intensities and to eliminate random noise especially from probe we confirmed that the cRNA samples were fragmented to uniform sets at the borderline of expression. Reproducibility of the nor- size (Fig. 1E) before continuing with sample labeling and hybrid- malized gene expression data was assessed by plotting gene ex- ization procedures. Gene expression profiling was performed on pression levels from each of the two replicate experiments (Fig. 1, these subsets by using CodeLink Uniset Human 20K I Bioarray. F–H). Linear correlation coefficient for all plots was above 0.98, The labeled cRNA samples for the three NK subsets were hybrid- and Ͻ0.01% of the genes examined displayed Ͼ2-fold variation in ized in duplicate to enhance the significance of the hybridization all three comparisons. This observation provided a justification to 6550 TRANSCRIPTOMES IN HUMAN NK SUBSETS

average normalized hybridization intensity values for each gene in between: 1) CD56brightCD16Ϫ vs CD56dimCD16ϩ; 2) activated replicate experiments, and to use these values for estimating gene CD16ϩ NK vs CD56dimCD16ϩ; 3) activated CD16ϩ NK vs expression patterns. The online version of this article contains sup- CD56brightCD16Ϫ. The online version of this article contains supplemental Table I that lists the averaged normalized hybridization plemental Table II that lists all genes and their changes in expres- values for the three NK subsets studied.8 sion calculated from the three comparisons mentioned above. Pair- A list of differentially expressed genes among NK cell subsets wise comparison of CD56brightCD16Ϫ and CD56dimCD16ϩ was compiled using an algorithm that identifies significant differ- revealed that, quantitatively, the difference in gene expression be- ences with minimal false positives (as estimated from an analysis tween these subsets (Fig. 2, C and D) was largely due to down- of marker genes that were included or excluded using different regulation of genes in CD56brightCD16Ϫ, rather than up-regula- thresholds as described in Materials and Methods). The NK sub- tion, as illustrated by a Venn diagram (Fig. 2, C and D). There sets were organized based on the overall similarity in gene expres- were 888 genes transcribed at significantly lower levels in the sion patterns by an unsupervised hierarchical clustering algorithm CD56brightCD16Ϫ subset, while 380 genes were specifically up- of variable genes that showed more than a relative 4-fold change regulated. Interestingly, CD56brightCD16Ϫ cells seemed to have in expression in at least one of the examined samples. A den- gene expression similarities with the activated CD16ϩ NK subset. dogram, in which the pattern of length of the branches reflects the One-hundred-thirty two genes of the total 380 up-regulated genes comparative difference in gene expression profiles between each of in CD56brightCD16Ϫ NK vs CD56dimCD16ϩ were also up-regu- the NK samples, is shown in Fig. 2A. Interestingly, activated lated, mostly at higher fold changes, in activated CD16ϩ NK when CD16ϩ NK cells were strikingly distinct from both subsets of un- compared with CD56dimCD16ϩ (Fig. 2C). In comparison, there ϩ activated pbNK, as illustrated by the length of the two terminal were no genes that were overexpressed in both CD56dimCD16 Downloaded from branches. Within the group of unactivated pbNK cells, a secondary and activated CD16ϩ NK compared with CD56brightCD16Ϫ NK. branching point separated CD56brightCD16Ϫ and CD56dimCD16ϩ A similar pattern was observed for down-regulated genes (Fig. duplicates from each other, thus confirming the fact that these two 2D). Taken together, this analysis suggests that the unactivated subsets are distinct. In other words, the gene expression profiles of low cytotoxic CD56brightCD16Ϫ NK subset has increased ex- the two subsets of unactivated NK cells were far more closely pressed levels of a number of activation-induced markers that can ϩ ϩ

related to each other than either was to activated CD16 NK cells also be found on activated CD16 NK. After classiﬁcation of se- http://www.jimmunol.org/ (Fig. 2A). lected genes into functional categories (Fig. 4), differentially ex- To conﬁrm the validity of our data and correlate with gene expressed genes were evaluated to determine whether these genes pression data in the literature, we compiled a list of hybridization could potentially contribute to the functional differences between intensity values of selected marker genes expected to be absent or these subsets and/or provide new insights on NK cell behavior. present in the NK subset in question (Fig. 2B). All samples did not

contain transcripts specific for monocytes, B, or T cells (CD14, bright Ϫ dim ϩ CD22, CD28, and CD4), emphasizing the purity of the cells used CD56 CD16 vs CD56 CD16 NK cells: molecules in the study. In correlation with our sorting criteria, CD16 mRNA involved in regulation of cytotoxicity expression was not detected in the CD56brightCD16Ϫ subset, while Previous studies of unactivated CD56dimCD16ϩ NK revealed that by guest on September 24, 2021 CD56 mRNA expression was significantly higher in this subset these cells are naturally more cytotoxic than unactivated compared with CD56dimCD16ϩ. In agreement with previous ob- CD56brightCD16Ϫ NK, due to higher levels of granzyme and per- servations, inhibitory receptor KIR3DL2 mRNA was preferen- forin expression (4, 9). Similarly, our analysis shows that effector tially expressed in unactivated CD56dimCD16ϩ NK cells (9), cytotoxic molecules granzyme B and CTLA-1 were expressed at while activating receptor KIR2DL4 mRNA was specifically tran- lower levels in the CD56brightCD16Ϫ subset (by ϳ2.2- and ϳ2- scribed in unactivated CD56brightCD16Ϫ and in vitro-activated fold, respectively) (Fig. 4C), while differences in granzymes A and CD16ϩ NK, but not unactivated CD56dimCD16ϩ NK (14). Our M were not statistically significant. Surprisingly however, gran- results also confirm L-selectin mRNA-specific expression on zyme K, that also has the ability to lyse target cells, was robustly CD56brightCD16Ϫ NK cells (6), preferential expression of gran- up-regulated in CD56brightCD16Ϫ NK (by ϳ20-fold) (Fig. 4C). zyme B in CD56dimCD16ϩ NK cells regardless of their activation This observation, together with the fact that CD56brightCD16Ϫ state (9), and specific expression of the NK lysis receptor NKp44 cells express adequate levels of other granzyme subtypes, led us to transcript following activation (15). Finally, it was important to investigate additional possibilities for their low cytotoxic ability. assess whether protein expression of the corresponding gene prod- We examined whether expression of adaptor proteins associated ucts of interest correlated with the transcriptional differences in with NCR and NKG2D activating receptors (such as DAP10, these genes. As shown in Fig. 3, detection of several membrane KARAP/DAP12, FC⑀R1␥, and CD3␨ chain) could provide a com- (Fig. 3, A–I) and secreted proteins (Fig. 3, J–K) by flow cytometry plementing explanation for this phenomenon. These molecules analysis, correlated with normalized hybridization intensities ob- contain ITAM or YxxM motifs in their cytoplasmic domains and tained for the same genes from our gene array analysis. Overall, are known to facilitate NK activation signals (16). The CD3␨ chain the concordance between the hybridization intensity values of the was down-regulated by Ͼ3-fold in CD56brightCD16Ϫ NK (Fig. various marker genes and published gene expression patterns in 4B). However, no significant differences in expression of other NK subsets validates our normalization methods and the quality of adaptor molecules were detected. The detection of CD3␨ protein NK samples used in this study. by intracellular flow cytometric staining consistently showed ϳ4- fold down-regulation in CD56brightCD16Ϫ NK in all donors tested Comparison of gene expression between unactivated (Fig. 5A). Recent evidence shows that the generally impaired T CD56brightCD16Ϫ and CD56dimCD16ϩ NK cells cell function in chronic inflammation results from down-regulation ␨ To begin the comparison between the various NK subsets, a list of the CD3 chain in vivo (while other TCR complex subunits was generated of differentially expressed genes by at least 2-fold remain preserved), thus reflecting the impact of this adaptor molecule on T cell functionality (13). As CD3␨ is the main signaling adaptor molecule for NKp30 and NKp46 cytotoxicity receptors Ϫ 8 The online version of this article contains supplemental material. (17), the relative deficiency in this molecule in CD56brightCD16 The Journal of Immunology 6551 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. CD56brightCD16Ϫ, CD56dimCD16ϩ, and activated CD16 cells represent three different NK cell subsets. A, Unsupervised hierarchical clustering of NK samples based on expression profile of genes with variable expression levels across all subset samples. Normalized data for genes that were up- or down- regulated by at least 4-fold in at least two of the samples were filtered and log transformed before cluster analysis. Mean levels for each gene across all samples were calculated and the magnitude of relative expression of a particular gene relative to the calculated mean expression was reflected by use of color representation. Brighter red means higher expression, brighter green means lower expression, and black means average intensity across samples. The organization and length of the branches in the resulting dendogram reflect the similarity in gene expression profiles between each of the samples. B, Expression levels for selected genes whose expression has been extensively characterized on NK subsets. C and D, Venn diagrams generated by the intersection of the list of genes up-regulated (C)or down-regulated (D) by at least 2-fold (based on selection criteria described in Materials and Methods) in at least one of the three comparisons performed: 1) CD56brightCD16Ϫ vs CD56dimCD16ϩ; 2) activated CD16ϩ NK vs CD56dimCD16ϩ; 3) activated CD16ϩ NK vs CD56brightCD16Ϫ. For example, a total of 380 genes were overexpressed in CD56brightCD16Ϫ when compared with CD56dimCD16ϩ (C), 216 of which were overexpressed in this paired comparison only, and not when activated CD16ϩ NK cells were compared with any of the two peripheral blood-derived unactivated NK subsets. Thirty-two genes were commonly up-regulated in all three comparisons performed. One-hundred thirty-two transcripts were up-regulated in CD56brightCD16Ϫ and activated CD16ϩ NK vs CD56dimCD16ϩ NK, while no genes were overexpressed in both CD56dimCD16ϩ and activated CD16ϩ NK vs CD56brightCD16Ϫ NK. 6552 TRANSCRIPTOMES IN HUMAN NK SUBSETS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Confirmation of expression patterns of selected differentially expressed genes at the protein level. CD56dimCD16ϩ, CD56brightCD16Ϫ, and activated CD16ϩ NK were sorted and purified as described in Materials and Methods. Each data set (A–K) shows flow cytometry staining for different molecules (light gray) compared with background staining (empty histograms). Dashed lines were added to assist in demonstrating the observed differences in protein expression. Mean fluorescence intensities (MFI) for each sample are indicated in the right upper corner of the panels. MFIs for background stainings were between 2 and 4. Bar graph above each histogram data set represents the normalized hybridization intensity of a probe set specific for the same gene. Data shown are representative stainings obtained from three to five different donors. The Journal of Immunology 6553 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Gene expression patterns in human CD56dimCD16ϩ and CD56brightCD16Ϫ NK subsets. Gene expression proﬁles of selected genes differentially expressed by at least 2-fold between CD56dimCD16ϩ and CD56brightCD16Ϫ subset. Bars represent fold change of the mRNA level of a particular gene when comparing these subpopulations. Positive values indicate that the transcript was more abundant in the CD56brightCD16Ϫ subpopulation and negative values indicate the opposite. Genes were grouped according to their presumed function (A–G) based on information available in public databases or in the literature. (Figure continues) 6554 TRANSCRIPTOMES IN HUMAN NK SUBSETS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. (Continued)

NK suggests an additional explanation for the lower cytotoxic ca- relates with the reconstitution of both CD3␨ and NKp30 on acti- pabilities of this subset. vated CD16Ϫ NK cells (Fig. 5, B and C) further strengthens our When examining differences in surface abundance of activating hypothesis regarding the involvement of these molecules in control- receptors on pbNK subsets, NKp30 was the only receptor found at ling CD56brightCD16Ϫ NK cytotoxicity. A pattern of enrichment was lower levels on the CD56brightCD16Ϫ NK subset (probes not rep- also observed for cytotoxicity costimulatory (enhancing) receptors on resented on the gene microarray used), while NKp46 and NKG2D CD56dimCD16ϩ NK. NK, T, and B cell Ag and CD58 (both belong surface expression on this subset was preserved (Fig. 5A). The to CD2 family), LLT1- lectin-like NK receptor (a member of NK observation that the impaired cytotoxic ability of CD56bright gene complex), and transmembrane protein 2 (TMEM2), all charac- CD16Ϫ NK cells was abolished following IL-2 activation and cor- terized as enhancers of NK cytotoxicity upon ligation (18–20), were The Journal of Immunology 6555 Downloaded from

FIGURE 5. Correlation between CD3␨ chain and NKp30 expression levels and CD16Ϫ NK cell cytotoxicity. A, Staining of isolated unactivated polyclonal pbNK cells for the CD3␨ chain (intracellular staining) and surface expression of NKp30, NKp46, and NKG2D receptor. Staining for CD56 was http://www.jimmunol.org/ used as a second marker to discriminate between CD56bright and CD56dim subsets. Mean fluorescence intensities for each subset are indicated. B, Staining of the activated polyclonal CD56ϩCD3Ϫ NK line for the CD3␨ chain (intracellular staining) and the NKp30 receptor. Staining for CD16 was used to differentiate between CD16ϩ and CD16Ϫ activated NK subsets. C, Killing of 721.221 cells by various purified NK subsets at various E:T ratios. Data are representative of three independent experiments. up-regulated on CD56dimCD16ϩ NK (Fig. 4A). CD2 was the gands. Specific expression of L-selectin on CD56brightCD16Ϫ NK only costimulatory receptor expressed at higher levels on the has provided a partial explanation for the ability of this subset to CD56brightCD16Ϫ NK subpopulation. To conclude this aspect traffic to secondary lymphoid organs (6). Our analysis confirms by guest on September 24, 2021 of the analysis, our data indicate that suppression and regulation this observation (Figs. 3A and 4A) and shows additional differ- of CD56brightCD16Ϫ NK killing might be a result of complex ences in adhesion molecule expression. CD58 (LFA-3), ICAM2, ␣ dim ϩ mechanisms in different hierarchies of the cytotoxicity activa- and integrin E were up-regulated in the CD56 CD16 subset, ␣ ␣ ␣ tion cascade that complement each other. These mechanisms while integrin 5, integrin M, integrin X, ICAM3, and CD44 involve down-regulation of certain activating (NKp30) and co- were up-regulated in the CD56brightCD16Ϫ NK subset (Fig. 4A). stimulatory receptors, key adaptor molecules (CD3␨), and cy- The CD44 protein has been implicated in lymphocyte trafficking totoxicity effector-secreted proteins (perforin, granzyme B, and to lymph nodes and enhancing IFN-␥ secretion (21, 22), two mo- CTLA1 molecules). dalities that CD56brightCD16Ϫ NK efficiently perform. The bright Ϫ bright Ϫ dim ϩ CD56 CD16 NK subset expresses CD44 in higher levels both CD56 CD16 vs CD56 CD16 NK cells: adhesion at the transcript level and at the cell surface (by ϳ3.75- and ϳ8- molecules ␣ fold, respectively) (Figs. 4A and 3B, respectively). Integrin E is The complex process of lymphocyte trafficking is dictated via in- considered an important factor for localizing certain lymphocyte teractions between cell surface adhesion molecules and their li- subsets to mucosal-epithelial tissues such as the gut and pancreatic

FIGURE 6. Unique expression of IL-7 receptor ␣-chain on CD56brightCD16- NK subset. A, Flow cytometry analysis of sorted CD56brightCD16Ϫ and CD56dimCD16ϩ NK subsets for IL-7R ␣-chain. B, Fold increase in proliferation of puriﬁed CD56brightCD16Ϫ and CD56dimCD16ϩ NK subsets in response to addition of 20 ng/ml recombinant human IL-7 for 72 h in 37°C. Data presented are representative of two independent experiments. 6556 TRANSCRIPTOMES IN HUMAN NK SUBSETS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. Gene expression patterns in human activated CD16ϩ and CD56dimCD16ϩ NK subsets. Gene expression proﬁles of selected genes differentially expressed by at least 2-fold between the unactivated CD56dimCD16ϩ and activated CD16ϩ NK subset. Bars represent the fold change of the mRNA level of a particular gene when comparing these two subpopulations. Positive values indicate that the transcript was more abundant in activated CD16ϩ NK and negative values indicate the opposite. The presented genes were grouped according to their presumed function (A–G) based on information available in public databases or in the literature. (Figure continues) The Journal of Immunology 6557 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. (Continued) 6558 TRANSCRIPTOMES IN HUMAN NK SUBSETS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 8. Characterization of functional interactions between activated CD16ϩ NK and T cells. A and B, Activated human CD16ϩ NK cells costimulate TCR responses of human CD3ϩ T cells. A, Proliferation of purified peripheral blood CD3ϩ T cells (50 ϫ 103 cells per well) in the presence of irradiated unactivated polyclonal NK cells or activated CD16ϩ NK cells (25 ϫ 103 cells per well) with or without suboptimal levels of anti-CD3 mAb (0.1 ␮g/ml). As a positive control, we used soluble anti-CD28 Ab (20 ng/ml) in addition to anti-CD3. Proliferation was (Figure legend continues) The Journal of Immunology 6559 islets (23, 24). Recent observations in mice showed increased NK- play an essential role in recruiting inflammatory effector cells to specific transcripts in pancreatic islets of type I diabetic mice and peripheral inflammation sites during murine CMV infection (30). that depletion of these cells inhibited diabetes development (25). Therefore, the specific abundance of these chemokines in Others demonstrated in rat models that gut-derived NK cells were CD56dimCD16ϩ NK correlates with the abundance of this subset equally cytotoxic to pbNK cells (26). In light of these observa- in inflamed tissues rather than secondary lymphoid organs. Lym- tions, human cytotoxic CD56dimCD16ϩ NK cells may preferen- photactin, which was also transcribed significantly in tially localize to gut-associated lymphoid tissue and play a role in CD56dimCD16ϩ NK and was also known to be important in lym- local immune surveillance. phoid recruitment to inflammatory tissues, was enriched in CD56brightCD16Ϫ (by ϳ4-fold, Fig. 4C). This chemokine has been bright Ϫ dim ϩ CD56 CD16 vs CD56 CD16 NK cells: implications for uniquely shown to have the capacity to modulate costimulatory NK development and homeostasis levels of human CD4ϩ and CD8ϩ T cells following TCR priming NK cell differentiation occurs when precursors interact with cyto- in lymph nodes (31), implying that lymphotactin secretion by Ϫ kines and stromal cells in the bone marrow (4). The up-regulation CD56brightCD16 NK cells present in T cell areas of secondary of IL-7R ␣-chain mRNA (by ϳ11-fold, Fig. 4A) and specific sur- lymphoid organs (6) might constitute a cross-talk pathway with face expression (Fig. 6A) on CD56brightCD16Ϫ NK cells, but not neighboring T cells. Ϫ CD56dimCD16ϩ NK, might also relate to certain developmental CD56brightCD16 NK expressed higher levels of lysozyme stages of this subset. Despite the low levels of IL-7R expression, mRNA (by ϳ5.6-fold, Fig. 4C), whose natural substrate is the this receptor was functional and induced specific proliferation in bacterial cell wall peptidoglycan (cleaving the ␤[1–4] glycosidic Ϫ CD56brightCD16 NK cells (Fig. 6B). This receptor is considered linkages between N-acetylmuramic acid and N-acetylglu- Downloaded from as one of the key players in maintaining homeostasis and survival cosamine), and granulysin which is a cytolytic granule member of naive and memory T cells (27), which raises the question of known to reduce the viability of a broad spectrum of intra- and ϩ whether it has a similar function in CD56brightCD16Ϫ NK ho- extracellular pathogens (32). CD56dimCD16 NK preferentially meostasis. Also, it has been shown in mice that the IL-7R is expressed defensin-6 (by ϳ5.8-fold) which is known to disrupt present on NK cell precursors; however, they lose its expression bacterial membranes (33). A secreted protein specifically up-reg- bright Ϫ upon terminal maturation and acquisition of cytotoxic capabilities ulated in CD56 CD16 NK cells (by ϳ6.9-fold) was amphi- http://www.jimmunol.org/ (11). Because it is not known yet whether mice have NK cell regulin, a member of the epidermal growth factor (EGF) family subsets analogous to CD56bright and CD56dim, data regarding hu- that interacts with the EGF/TGF-␣ receptor to promote the growth man NK subset development has been limited. Nevertheless, based of normal epithelial cells and inhibits the growth of certain ag- on the inverse correlation between maturation/cytotoxic ability and gressive carcinoma cell lines (34). These observations suggest that IL-7R expression in the mouse NK cell lineage, our results can be highly purified NK cells might posses additional novel and direct interpreted in a way that supports a current hypothesis regarding cytotoxic pathways against invading microorganisms or cancerous human NK subset development, claiming that CD56dim cells are cells by secreting certain natural antimicrobial or growth-suppress- derived from the CD56bright NK subset following further matura- ing agents. tion (4). The proinflammatory cytokine, osteopontin, has been shown to by guest on September 24, 2021 The interaction between Notch and its ligands, delta or jagged critically aggravate development and progression of experimental family members, constitutes an evolutionary conserved pathway autoimmune encephalomyelitis (EAE) in mice through enhancing for determining cellular development pathways (28). Notch sig- Th1 activity (35). Osteopontin was specifically transcribed in ϩ naling is intimately involved in the process of T vs B lymphocyte CD56dimCD16 NK cells (Fig. 4C). Interestingly, these cells have differentiation from common progenitor precursors (28). More- been shown to infiltrate inflammatory lesions in CNS, especially at over, Notch1 signaling has been shown to attenuate peripheral T peak stages in the development of the disease (36). This observa- cell activation by inhibiting activation and cytokine production tion calls for further evaluation of osteopontin production by this (29). Our analysis yielded significant levels of Notch1 and Notch2 NK subpopulation in EAE models and exploring direct and indi- transcripts in CD56dimCD16ϩ and, more abundantly, in rect links between these cells and autoreactive T and B cells. CD56brightCD16Ϫ NK cells (Fig. 4A) thus suggesting possible in- bright Ϫ dim ϩ volvement of this pathway in regulating NK subset development, CD56 CD16 vs CD56 CD16 NK cells: other differences function, and homeostasis. As mentioned earlier, one aim of this study was to analyze certain

bright Ϫ .dim ϩ members of the group of proteins that were up-regulated both in CD56 CD16 vs CD56 CD16 NK cells: secreted CD56brightCD16Ϫ and activated CD16ϩ NK cells compared with immune effector molecules the CD56dimCD16ϩ subset (Fig. 2C). Interestingly, several of the Differentially expressed genes coding for secreted proteins, such as genes in this group belong to the MHC class II family (HLA-DRA, chemokines, were studied. It was found that CD56dimCD16ϩ NK HLA-DRB, HLA-DMA, HLA-DPA, HLA-DQA, HLA-DPB, and expressed signiﬁcantly higher levels of IL-8, MIP-1␣, MIP-1␤, CD74) (Figs. 4A and 3C). This expression pattern suggests the and RANTES transcripts (Fig. 4C). This group of chemokines has functional possibility that superantigens may bind to MHC class been demonstrated to be expressed in mouse NK cells in vivo, and II-positive CD56brightCD16Ϫ or activated NK cells in lymph nodes

determined by [3H]thymidine incorporation after 72 h of total incubation time. B, Before cell harvesting, 100 ␮l of supernatant from each well was removed for ELISA measurement of IL-2. Values are mean Ϯ SD for triplicate samples. C, A total of 1 ϫ 106 unactivated polyclonal CD56ϩCD3Ϫ NK cells were grown for 7 days at a concentration of 0.5 ϫ 106/ml in RPMI 1640 medium ϩ 10% heat inactivated FCS with or without the addition of 100 U/ml IL-2 and 1 ng/ml PHA. Subsequently, we removed supernatants of these cultures and introduced them to Jurkat cells at a concentration of 1 ϫ 106/ml for 8 h. Incubation of Jurkat cells with control medium that contained IL-2 and PHA was performed as a control to rule out that the apoptotic effect observed was a result of activation-induced cell death. Jurkat cells were then analyzed for apoptosis levels by flow cytometry. Propidium iodide-positive (necrotic) cells were excluded from the analysis, while the remaining cells were characterized for Annexin V staining percentages. All experiments shown are representative samples of three total repeats performed. 6560 TRANSCRIPTOMES IN HUMAN NK SUBSETS or peripheral inflammatory tissues and potentiate the pathologic progression and proliferation (e.g., CDC2, CDK4, CCNA2, and nonspecific T cell proliferation and activation. CCNB2, CCNA2, MCM2, CKS2 etc) (Fig. 7F and data not Lymphotoxin ␤ is a type II membrane protein of the TNF fam- shown) were robustly, and in most cases, specifically expressed in Ϫ ily. It was uniquely transcribed in CD56brightCD16 NK cells (up- the highly proliferating activated CD16ϩ NK cell subset, while regulated by ϳ8-fold, Fig. 4A), and robustly overexpressed after antiproliferative genes BTG1 and cyclin dependent kinase inhibi- ϩ activation on CD16 NK cells (up-regulated by ϳ50-fold, Fig. tor 1C were down-regulated. We then focused on selected differ- ␣ ϩ 7A). This protein anchors lymphotoxin to the cell surface entially expressed genes between CD56dimCD16 and activated through a heterotrimer complex that has the ability to bind the CD16ϩ NK subsets, and classified them into functional categories ␤ ␤ lymphotoxin receptor (37). Surface expression of lymphotoxin (Fig. 7). on B cells in secondary lymphoid organs has a crucial role in supporting correct lymphoid architecture that is critical for an ef- bright Ϫ fective immune response (37). The fact that CD56 CD16 NK Activated vs unactivated CD16ϩ NK cells: molecules involved in cells are preferentially found in lymph nodes might supply the the regulation of non-NK immune functions biological rationale for specific expression of lymphotoxin ␤ on this subset. In contrast, this molecule is an inducer of proinflam- The naive T cell responses to Ag are directed by immunogenic matory cytokines and chemokines in effector cells localizing to stimuli that can be divided into two main categories. First, TCR inflamed tissues (38). It is possible that activated CD16ϩ NK use ligation mediated by peptide-MHC complexes expressed on pro- this robust induction in lymphotoxin ␤ to contribute to the pro- fessional APCs. Second, costimulatory signal conferred by a gression and potentiation of the ongoing immune response. counter-receptor expressed on APC or other accessory cells (42), Downloaded from Ϫ CD56brightCD16 NK cells have been shown to be generally which is mandatory for efficient priming of the immune response better responders to various cytokine stimulations (response can be and for avoiding anergy (43). OX40L, CD70, and CD86 which are observed at relatively lower concentrations of cytokine stimula- ligands for the classical TCR costimulatory molecules OX40, dim ϩ tion), as compared with the CD56 CD16 NK subset, due to CD27, and CD28, respectively (43), were exclusively transcribed preferential expression of a number of membrane-associated re- in activated NK cells (by ϳ2.5-, 4-, and 6-fold, respectively) (Fig. ceptors (e.g., c-kit receptor, high affinity IL-2R, etc.) (39). The 7A). The observed transcriptional pattern for these molecules was http://www.jimmunol.org/ results indicated that several proteins expressed at higher levels on verified by detecting cell surface protein expression (Fig. 3, G–I). the CD56bright Ϫ - CD16 NK subset could also contribute to the en Class IV semaphorin Sema4A, usually expressed on DCs and hanced cytokine responsiveness of this subset: 1) ITAM contain- known to costimulate T cell responses (44), was also transcribed in ing signal transducing adaptor molecule STAM (by ϳ2.5-fold, all three NK subsets examined at similar levels (data not shown). Fig. 4B) that is known to associate with JAK2 and JAK3 kinases We extend the relevance of these observations by showing that and participates in downstream signaling of the cytokine receptor ϩ upon phosphorylation (40); 2) SRY-box 4 (SOX4) transcription irradiated activated CD16 NK cells were extremely potent in ϳ costimulating proliferation and IL-2 secretion of peripheral blood factor (by 10-fold, Fig. 4D) that is involved in controlling cyto- ϩ kine induced cellular transcriptional regulation (41); 3) ribosomal CD3 T cells that were activated by suboptimal levels of plate- by guest on September 24, 2021 protein family members L36, L35, L27A, L3, L22, L10A, and S26 bound anti-CD3 mAb (Fig. 8, A and B). Although it is possible that (Fig. 4G) that catalyze protein synthesis were also preferentially the irradiated activated NK cells used can produce part of the IL-2 expressed in CD56brightCD16Ϫ NK. This observation might sug- detected in this experiment, direct intracellular staining of T cells gest that this subset is more “metabolically ready” to engage in the confirmed the up-regulation of IL-2 expression upon incubation ϩ protein synthesis process that is required for functional respon- with anti-CD3 and activated CD16 NK cells (data not shown). siveness to cytokines. The presence of anti-CD3 mAb, in addition to activated NK cells, Lastly, many other genes overexpressed in one of the unacti- was mandatory for obtaining robust T cell proliferation further vated pbNK subsets that are presented in Fig. 4 or in on-line sup- strengthening the conclusion that TCR costimulation enhanced plementary Table II (e.g., EBV-induced gene (EBI2), VEGF␤, CD3ϩ T cell activation in this experiment (Fig. 8, A and B). It is lymphopain, TCF7, annexin A2, CD55, etc.) are of considerable important to note that OX40-OX40L interaction has been de- interest but are beyond the scope of the present paper. The data scribed as a major pathway in maintaining long-term survival of presented in these figures should be useful to those interested in neutrophils and primed T cells in vivo (45). Taken together, these analyzing differentially expressed genes that were not discussed in results provide direct evidence for novel and unexpected cross-talk this manuscript. pathways between NK and T cells as TCR costimulators, linking Characterization of CD16ϩ NK cell transcriptome following the innate and adaptive immune responses. activation The above observations encouraged us to search for other molecules on NK cells that might mediate direct interaction with im- To gain more insight into potential functions that NK cells might mune cells. Tim3 (T cell Ig- and mucin-domain-containing mole- exert upon activation in inflamed tissues, we analyzed transcrip- ϩ ϩ cule) was significantly induced on activated CD16 NK cells (by tional changes in CD16 NK cells following activation. Activated ϩ ϳ3-fold, Fig. 7A). This molecule is known to be expressed on Th1, CD16 NK cells analyzed in this work were activated with a com- ϩ bination of stimuli including IL-2, PHA, cytokines produced by but not Th2, CD4 T cells (46), where it is thought to down- irradiated nonautologous PBLs (used as feeders), and addition of regulate macrophage homeostasis and activation levels and auto- classical NK target cells (irradiated RPMI 8866 cells). Therefore, regulate the severity of Th1-mediated immune responses (46, 47). it was not surprising to observe the dramatic alteration in gene Additionally, as Th1 and Th2 cells negatively regulate each other’s expression profile in the activated CD16ϩ NK cells (Fig. 2, A, C, functions, Th1 cells might be implicated in promoting asthma and and D). atopy (typical Th2 immune responses) via Tim3-Tim3 ligand in- Initially, genes up-regulated in the activated CD16ϩ NK line teractions (48). We therefore speculate that NK cells might also compared with both pbNK subsets (Fig. 2C) were evaluated. Ex- influence macrophage behavior and participate in balancing Th1- pectedly, a group of over 30 genes known as inducers of cell cycle Th2 cross-regulation. The Journal of Immunology 6561

Activated vs unactivated CD16ϩ NK cells: alterations in tivated, but not resting, CD4ϩ and CD8ϩ T cells. This would lead trafficking capabilities to attenuation of T cell activation and resolution of the immune response (60). Galectin 3 was another family member up-regulated Chemokines and chemokine receptors are pivotal players in traf- ϩ ficking, homing, and retention of immune and nonimmune cells. in CD16 NK cells following activation (Figs. 7C and 3K). It was The activity of several chemokines is regulated by CD26 (dipep- shown previously that intracellular galectin-3 induction following tidyl-peptidase IV)-mediated cleavage (49). The results indicate activation of B cells is important for long-term survival of these that mRNA encoding CD26 was uniquely transcribed in activated cells during the immune response (61). Studies are currently eval- CD16ϩ NK and up-regulated by Ͼ12-fold (Fig. 7A). The inacti- uating whether galectin 3 has a similar effect on activated NK cell vation of chemokines by CD26, together with down-regulation of survival and homeostasis. CXCR4 (Fig. 7A), may contribute to the fine control of chemo- ϩ tactic migration of these cells by providing a “stop” signal that Activated vs unactivated CD16 NK cells: other differences keeps these cells at the site of inflammation. In correlation with the enhanced cytotoxic activity of activated The cell surface molecules: CD9, CD53, CD63, CD81, and CD16ϩ NK cells, the lysis inhibitory receptor NKG2A and the CD151, all belonging to the tetraspanin family (also known as orphan NKRp1 lectin-like receptor were down-regulated upon NK ϩ transmembrane 4 superfamily), were up-regulated on CD16 NK activation (Fig. 7A). In contrast, NKG2C and NKG2E (KLRC2 cells after activation (Fig. 7A). Flow cytometry analysis confirmed and KLRC3) were up-regulated on activated NK cells (Fig. 7A). CD9, CD53, and CD81 up-regulation on this NK subset (Fig. 3, Both receptors are CD94-associated members of the C-type lectin D–F). Many of the tetraspanins assemble with various integrin like receptor family and lack an ITIM in their cytoplasmic domain. Downloaded from subunits into functional signaling complexes and facilitate alter- NKG2C associates with DAP12 and acquires activating functions. ation in cell-cell and cell-matrix interactions (50–53). Thus, over- It is presumed that, due to the structural similarities between these expression of certain tetraspanin family members on activated NK two receptors, NKG2E might also facilitate activating functions. cells might underlie mechanisms used by these cells to enhance or However, the ligands and function of NKG2E have not been char- alter their migration and retention in inflamed tissues. Addition- acterized so far. Up-regulation of the NK costimulatory molecules ally, CD81 is known to confer inhibition of NK cell proliferation CD2 and CD59, together with the Fc⑀RI␥ chain on activated NK (54). Interestingly, the IFN-induced transmembrane proteins 1 and cells might also contribute to the lytic potency of these cells http://www.jimmunol.org/ 3 (IFITM1 and IFITM3) that are known to be involved in prevent- (Fig. 7A). ing overproliferation of T cells (55) were also up-regulated on Many transcripts induced or down-regulated in CD16ϩ NK cells ϩ CD16 NK cells following activation (Fig. 7A). This suggests that upon activation are presented in Fig. 7 or in on-line supplementary NK cells, similarly to T cells, might use the up-regulation of these Table II (e.g., TRAIL, CLECSF2, SIGLEC7, CD55, dopamine D4 receptors together with CD81 as part of an autoregulatory “brake” receptor, several G protein coupled receptors, MMP25, survivin, mechanism that controls their activation-induced proliferation. etc.) and are of considerable interest, but are beyond the scope of the analysis presented in this paper. ϩ

Activated vs unactivated CD16 NK cells: secreted immune by guest on September 24, 2021 effector molecules Discussion In agreement with previous observations, granzyme A, granzyme The morphological analysis of changes in the phenotypic charac- B, CTLA1, and granzyme K were up-regulated on activated teristics of various lymphocyte subsets is still the basis for dissect- CD16ϩ NK, while granzyme M displayed a unique transcription ing functional pathways underlying the biological properties of pattern compared with other members of the granzyme family both normal and malignant cells. Despite the challenges posed by (down-regulated by ϳ2.5-fold upon activation; Fig. 7C). Gran- our genome size, large scale expression analysis in humans has zyme M is known to be exceptional in that its expression levels been proven to be overwhelmingly productive. Increasing genome have been previously shown not to correlate with the cytotoxic sequence information for different organisms, development of ability of NK cells. Granzyme M also induces cell death by a powerful robots for arraying, and the availability of widely acces- unique mechanism not featuring DNA fragmentation and occur- sible “user-friendly” tools for systematical handling of the ring independently of caspases (56, 57). genomic analysis output have all accelerated the use of microar- Galectins, also referred to as S-type lectins, are a conserved rays in basic and clinical research (62). The discrepancies observed family of proteins defined by the presence of at least one charac- between gene expression and protein abundance suggest that post- teristic carbohydrate recognition domain (58). Activated CD16ϩ translational modalities may be at least as important as changes in NK cells up-regulated expression of galectin 1 and galectin 12 mRNA levels in determining the cellular protein composition and (Fig. 7C), both characterized as inducers of apoptosis (59). Galec- provide a cautionary note for efforts to interpret cell composition tin 1 expression at the protein level on activated CD16ϩ NK cells and function in relation to mRNA levels only (63). Still however, was verified by intracellular flow cytometry staining (Fig. 3J). This transcriptional analysis can be highly efficient in providing initial protein has been shown to mediate apoptosis of peripheral acti- novel scientific leads and ideas that subsequently need to be re- vated T cells by segregating O-glycosylated CD45 from CD7 and evaluated and established at the protein and functional levels. CD43 (58, 59). Interestingly, supernatants of activated CD16ϩ NK In an effort to better characterize functionalities of NK subsets, cultures were able to induce low, but significant, levels of apopto- we have conducted a detailed characterization of gene expression sis of the CD7ϩ Jurkat T cell line (Fig. 8C). This effect was not a in highly purified conventional unactivated peripheral blood-de- result of IL-2 and PHA induced cell death, because addition of rived NK subsets and in vitro-activated CD16ϩ NK cells. Overall, control medium containing these materials did not induce such the global comparison between CD56brightCD16Ϫ and CD56dim apoptosis. This experiment strengthens the possibility of proapop- CD16ϩ unactivated NK subsets supports a model whereby these totic factor production by CD16ϩ NK cells following activation. subpopulations represent functionally distinct subsets of mature One hypothesis may be that induction of activated T cell apoptosis human NK cells. We present new data potentially underlying func- via NK-derived galectin-1 and 12 might act in concert with the tional differences between CD56brightCD16Ϫ and CD56dimCD16ϩ described ability of syngeneic NK cells to recognize and kill ac- NK subsets involving cytotoxicity induction, trafficking abilities, 6562 TRANSCRIPTOMES IN HUMAN NK SUBSETS homeostasis, and interaction with their microenvironment. Gener- similarly up-regulated on peripheral blood-activated NK cells in ally, the data support the notion that CD56brightCD16Ϫ NK cells our analysis (Fig. 7). One explanation for the activated-like phe- are regulatory cells that can be heavily involved in interacting with notype of the decidual NK subset can be inferred from the chronic neighboring immunocompetent cells found in lymphoid tissues. In intimate interaction of these cells with semiallogeneic extravillous contrast, CD56dimCD16ϩ NK cells seem to be skewed toward trophoblasts that invade maternal decidua and the cytokine-en- homing to inflammation sites and promoting immune responses, in riched local microenvironment (7, 64). Such conditions might in- addition to induction of cytotoxicity. duce, at least, a partial activation state on NK cells found in the Upon activation, CD16ϩ NK cells acquired diverse immuno- decidua. However, this raises the question regarding what is sup- regulatory activities together with enhanced cytotoxicity. Of inter- pressing decidual NK cells from secreting IFN-␥ and inducing est was an array of membrane and secreted molecules induced on cytotoxicity in vivo, despite the expression of these activation CD16ϩ NK cells following activation that highlight the role for markers? Recent observations in mouse models demonstrate de- lymphocyte-lymphocyte interactions in driving immune responses cidual and systemic in vivo expansion of maternal regulatory (sup- (Tim3, TCR costimulatory molecules, galectin family members, pressor) CD4ϩCD25ϩ T cells that suppress immune responses to- etc.). However, it might seem puzzling that activated CD16ϩ NK ward the fetus (66). Depletion of this subset led to a failure of cells can use opposing positive and negative pathways for influ- gestation due to immunological rejection of the fetus. In light of encing T and other lymphocyte subsets (costimulation of TCR re- our observation regarding various NK T cell cross-talk abilities, sponses vs induction of apoptosis and NKG2D-mediated killing of and the fact that regulatory CD4ϩCD25ϩ T cells can suppress activated T cells). We suggest two explanations for this phenom- non-T cell effectors (e.g., DCs) (67), it is possible that regulatory ϩ enon. First, the activated CD16 NK cells used in our array anal- T and NK cell interactions might underlie the suppression of de- Downloaded from ysis were subjected to multiple activating stimuli for two weeks cidual NK cell lytic activities. including IL-2, PHA, cytokines produced by irradiated feeder cells The data presented here suggest that human NK cells’ functions derived from two different donors (thus forming mixed lympho- extend beyond simple cytotoxicity induction by granzyme and per- cyte reaction conditions), and addition of irradiated target cells that forin secretion, and that modulation of the immune response is not cross-link several NK activating and costimulatory receptors. It is a preserved function for the CD56brightCD16Ϫ NK subset. Highly ϩ likely that different stimuli induce distinct signaling pathways each cytotoxic activated CD16 NK cells also have their unique abil- http://www.jimmunol.org/ leading to induction of certain functionally related protein groups. ities in influencing events occurring in their microenvironment. Therefore, the robust diversity in transcripts and proteins ex- The dynamic and complex transcriptional patterns in human NK pressed on activated CD16ϩ cells used in this study might have subsets presented and highlighted here constitute important candi- resulted from the combination of activating stimuli used. Second, dates for future in-depth functional studies. we hypothesize that molecules promoting activation and propaga- tion of the immune response are generally induced early in acti- Acknowledgments vation, while genes known to attenuate immune response and pre- We thank Nabil Hanna, Gayda Hanna, and Marilyn Kehry for helpful ideas vent hyperactivation “kick-in” at later stages. Several relevant and discussions. We also thank Nathan Regimbal and Ilan Vaknin for examples from T cell biology support the rationale behind this excellent assistance with cell sorting and flow cytometry. by guest on September 24, 2021 suggestion. Tim3, known to inhibit macrophage activation and de- ϩ References tected on activated CD16 NK (Fig. 7A), is induced on Th1 cells 1. Orange, J. S., M. S. Fassett, L. A. Koopman, J. E. Boyson, and J. L. Strominger. grown in Th1-polarizing conditions only after two rounds of re- 2002. Viral evasion of natural killer cells. Nat. Immunol. 3:1006. stimulation with the appropriate cytokines (47). Another example 2. Mandelboim, O., N. Lieberman, M. Lev, L. Paul, T. I. Arnon, Y. Bushkin, D. M. Davis, J. L. Strominger, J. W. Yewdell, and A. Porgador. 2001. 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Corrections

Hanna, J., P. Bechtel, Y. Zhai, F. Youssef, K. McLachlan, and O. Mandelboim. 2004. Novel insights on human NK cells’ immunological modalities revealed by gene expression proﬁling. J. Immunol. 173: 6547–6563

During the preparation of Fig. 3G, a flow cytometry analysis (FACS) plot image was inadvertently duplicated from Fig. 3D (middle panel). We now enclose a corrected Fig. 3 with the validated Fig. 3G. This change does not affect the conclusions or interpretations of findings presented in our article in any way. The figure legend was correct as published and is shown below for reference.

FIGURE 3. Confirmation of expression patterns of selected differentially expressed genes at the protein level. CD56dimCD161, CD56brightCD16–, and activated CD161 NK were sorted and purified as described in Materials and Methods. Each data set (A–K) shows flow cytometry staining for different molecules (light gray) compared with background staining (empty histograms). Dashed lines were added to assist in demonstrating the observed differences in protein expression. Mean fluorescence intensities (MFI) for each sample are indicated in the right upper corner of the panels. MFIs for background stainings were between 2 and 4. Bar graph above each histogram data set represents the normalized hybridization intensity of a probe set specific for the same gene. Data shown are representative stainings obtained from three to five different donors. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1590001