The Journal of Immunology

Evidence for NK Cell Subsets Based on Receptor Expression1

Robert D. Berahovich,2* Nu L. Lai,* Zheng Wei,* Lewis L. Lanier,† and Thomas J. Schall*

To help understand the role of in NK cell trafficking, we determined the profiles of three different human NK cell lines and freshly isolated primary human NK cells. The cell lines overlapped in their chemokine receptor profiles: CXCR3 and CXCR4 were expressed by all three lines, whereas CCR1, CCR4, CCR6, CCR7, and CX3CR1 were expressed by only one or two of the lines, and no other chemokine receptors were detected. Freshly isolated primary NK cells were found to express CXCR1, CXCR3, and CXCR4, and to contain subsets expressing CCR1, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR5, and CXCR6. With the exception of CCR4, these chemokine receptors were expressed at higher percentages by CD56bright NK cells than by CD56dim NK cells. In particular, CCR7 was expressed by almost all CD56bright NK cells but was not detected on CD56dim NK cells. CCR9 and CXCR6 have not been described previously on primary NK cells. These results indicate that within both the CD56bright and CD56dim NK cell populations, subsets with the capacity for differential trafficking programs exist, which likely influence their functions in innate and adaptive immunity. The Journal of Immunology, 2006, 177: 7833–7840.

atural killer cells are that participate in both CD56brightCD16Ϫ cells are a separate lineage of NK cells than innate and adaptive immune responses to malignant tu- CD56dimCD16ϩ cells. For example, CD56brightCD16Ϫ cells isolated N mors, intracellular pathogens, and foreign cells. NK from up-regulated perforin and become cytotoxic after 1 cells circulate in peripheral blood, but rapidly move to sites of wk of culture in medium containing IL-2 (6). In contrast, upon entry immune reactions in peripheral tissues, due to gradients of che- of CD56dimCD16ϩ NK cells into peripheral tissues, CD56 was up- mokines and lysolipids produced in adjacent capillaries (1–3). At regulated (3). It is likely that peripheral blood CD56brightCD16Ϫ NK these sites, NK cells recognize target cells via ligation of a variety cells are a heterogeneous population, potentially containing at least of activating receptors. Once activated, NK cells lyse the offending three different subsets: 1) immature cells coming from the bone mar- ␥ cells and release , including IFN- , which recruit other row, 2) mature cells activated in the lymph nodes, and 3) cell types and modulate subsequent responses. CD56dimCD16ϩ-derived cells returning to the bloodstream af- In human blood, two types of NK cells are found, based on ter activation in peripheral tissues. In addition, a subset of dim ϩ expression of CD56 and CD16: CD56 CD16 cells, which ac- CD56brightCD16Ϫ NK cells was found to be activated (e.g., ϳ bright Ϫ count for 90% of the NK cells, and CD56 CD16 cells, HLA-DRϩCD45ROϩ) (7). which comprise the remaining ϳ10% of the NK cells. CD56dim There are also inconsistencies in the literature regarding the che- CD16ϩ cells are the more cytotoxic population, whereas CD56bright mokine receptors that regulate NK cell trafficking (3). Many lab- CD16Ϫ cells express the high-affinity IL-2R CD25 and thereby pro- oratories analyzed IL-2-activated NK cells, sometimes with con- liferate to a greater extent after exposure to IL-2 and typically produce Ϫ taminating T cells, and most laboratories did not separate the greater amounts of cytokines (3). CD56brightCD16 cells are also CD56brightCD16Ϫ and CD56dimCD16ϩ NK cells. In the two most found at low frequencies in secondary lymphoid organs, e.g., lymph node and tonsil, and respond vigorously to locally produced IL-2 (4). complete analyses of fresh NK cells (8, 9), discrepancies were CD56brightCD16Ϫ cells have also been found in inflammatory lesions observed with regard to no less than five chemokine receptors. obtained from a wide variety of diseases (5). Therefore, we sought to re-evaluate the chemokine receptors on At present, the relationship between the two populations of NK freshly isolated NK cells by using flow cytometry and cells is controversial. Evidence exists that CD56brightCD16Ϫ cells are assays. Moreover, we used these methods to determine the che- direct precursors of CD56dimCD16ϩ cells; that CD56brightCD16Ϫ mokine receptor profile on three commonly used NK cell lines: cells are direct descendents of CD56dimCD16ϩ cells; or that NKL, KHYG-1, and NK-92. The NKL cell line was isolated from peripheral blood of a patient with CD56ϩCD16ϩ large granular leukemia (10). The KHYG-1 cell line was isolated *ChemoCentryx, Mountain View, CA 94043; and †Department of Microbiology and from peripheral blood of a patient with an aggressive NK leu- Immunology and the Cancer Research Institute, University of California San Fran- kemia (11). The NK-92 cell line was isolated from peripheral cisco, San Francisco, CA 94143 blood of a non-Hodgkin’s lymphoma patient containing large Received for publication January 6, 2006. Accepted for publication September 12, 2006. granular lymphocytes detected in the (12). The NK lines were used, in part, because they might be derived from The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance rare NK subsets and, as such, might express chemokine recep- with 18 U.S.C. Section 1734 solely to indicate this fact. tors not expressed on a substantial fraction of peripheral blood 1 This work was supported in part by National Institutes of Health Grants AI068129 NK cells. In this study, we present a comprehensive analysis of and AI056690. L.L.L. is an American Cancer Society Research Professor. chemokine receptor expression and function on these three cell 2 Address correspondence and reprint requests to Dr. Robert Berahovich, Chemo- Centryx, 850 Maude Avenue, Mountain View, CA 94043. E-mail address: lines, and a more selective analysis of freshly isolated human [email protected] NK cells.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 7834 CHEMOKINE RECEPTORS ON NK CELLS

Materials and Methods Cells Human buffy coats were purchased from the Stanford Blood Center. PBMC were prepared by centrifugation of the buffy coats over Ficoll- Hypaque (Sigma-Aldrich) for 45 min, then collecting and rinsing the cells at the interface with PBS. NK cells used in chemotaxis assays were pre- pared by negative depletion of the PBMC, using a commercial (Miltenyi Biotec) and an AutoMACS instrument (Miltenyi Biotec). NKL cells were a gift from M. Robertson (University of Indianapolis, IN) and cultured in RPMI 1640 (Mediatech), 10% FBS (HyClone), 10 mM HEPES (Mediatech), 1 mM sodium pyruvate (Mediatech), 1ϫ nonessential amino acids (Mediatech), 0.1 mM 2-ME (Sigma-Aldrich), and 200 U/ml IL-2 (R&D FIGURE 1. Expression of CD56 and CD16 on NK cell lines. NKL, Systems). KHYG-1 cells were purchased from the Health Science Re- KHYG-1, and NK-92 cells were analyzed by flow cytometry for surface search Resources Bank and cultured in RPMI 1640, 10% FBS, and 100 expression of CD3, CD16, and CD56. Top, Expression of CD3 vs CD56. U/ml IL-2. NK-92 cells were purchased from the American Type Culture Bottom, Staining by anti-CD16 Ab (filled histogram) vs isotype-matched Collection and cultured in Alpha MEM without ribonucleosides and de- control Ab (open histogram). oxyribonucleosides (Mediatech), 12.5% horse serum (HyClone), 12.5% FBS, 2 mM L-glutamine (Mediatech), 1.5 g/L sodium bicarbonate (Medi- atech), 0.2 mM inositol (Sigma-Aldrich), 0.02 mM folic acid (Sigma- Aldrich), 0.1 mM 2-ME, and 100 U/ml IL-2 (R&D Systems). using the CyQuant cell proliferation assay kit (Molecular Probes) and were analyzed with a Tecan fluorometer (excitation at 480 nm, emission at 530 Flow cytometry nm). Data were analyzed and plotted in arbitrary units of fluorescence using Prism (GraphPad Software). PBMC were exposed to human IgG at 50 ␮g/ml in buffer (PBS containing 5% FBS) on ice for 15 min to reduce nonspecific binding in subsequent steps. Cells were rinsed with buffer and exposed to anti-chemokine recep- Results tor Abs or isotype-matched control Abs (all purchased from R&D Systems) Determination of chemokine receptor expression on NK cell at 10 ␮g/ml in buffer on ice for 30 min. Cells were rinsed with buffer and lines Ј exposed to PE-conjugated F(ab )2 of anti-mouse IgG Ab (Beckman Coulter) on ice for 20 min. Cells were rinsed with buffer and exposed to 5% Flow cytometric analysis of the NKL, KHYG-1, and NK-92 cell normal mouse serum (Sigma-Aldrich) in buffer on ice for 15 min and lines with Abs specific for CD3, CD56, and CD16 indicated that rinsed again. Cells were finally exposed to a mixture of CyChrome-con- the lines were uniformly CD3 negative, but varied in CD56 and jugated anti-CD16, Alexa 647-conjugated anti-CD56, and FITC-conju- CD16 expression: NKL cells were CD56Ϫ/dimCD16ϩ, KHYG-1 gated anti-CD3, -CD14, and -CD19 Abs (BD Biosciences) in buffer on ice ϩ Ϫ bright Ϫ for 20 min. After rinsing with buffer, cells were analyzed on a FACScan cells were CD56 CD16 , and NK-92 cells were CD56 CD16 (BD Biosciences). NK cell lines were analyzed using the chemokine re- (Fig. 1). These results are in accordance with the previously published ceptor Abs and PE-conjugated anti-mouse IgG Ab described above. data (10–12). Each cell line was analyzed for chemokine receptor expression Chemotaxis assay by two methods: flow cytometric staining with Abs specific for NK cell lines and enriched NK cells were resuspended in HBSS containing chemokine receptors and chemotaxis assays with ligands specific 0.1% BSA. Chemotaxis assays were performed in 96-well ChemoTx mi- for chemokine receptors. NKL cells were found by flow cytometry croplates (Neuroprobe) as follows. Chemokines (Table I) were added to the lower wells (final volume 29 ␮l), and 20 ␮l of cell suspension (5 ϫ 106 to express CCR4, CCR7, CXCR3, and CXCR4; in contrast, CCR1, cells/ml) was added to the polycarbonate filter (5-␮m pore size). After CCR2, CCR5, CCR6, CCR8, CCR9, CXCR1, CXCR2, CXCR5, incubation at 37°C in a humidified environment for 2 h, the filters were and CXCR6 were not detected (Fig. 2A). In chemotaxis assays removed. Cells that migrated into the lower chamber were quantified by analyzing responsiveness to three concentrations of chemokines, ligands specific for CCR4 (CCL22/MDC), CCR7 (CCL19/MIP- 3␤), and CXCR4 (CXCL12/SDF-1␣) induced migration of the Table I. Chemokines used in this study NKL cells, whereas ligands specific for CCR1 and CCR5 (CCL3/ MIP-1␣), CCR2 (CCL2/MCP-1), CCR3 (CCL11/eotaxin), CCR5 Receptor Ligand Common Name (CCL4/MIP-1␤), CCR6 (CCL20/MIP-3␣), CCR8 (CCL1/I-309), CCR9 (CCL25/TECK), CXCR1 and CXCR2 (CXCL8/IL-8), CCR1 CCL15 Leukotactin CXCR3 (CXCL11/I-TAC), CXCR5 (CXCL13/BCL), and CXCR6 CCL23 CK␤8⌬24 CCR1,5 CCL3 MIP-1␣ (CXCL16) failed to induce migration of the NKL cells (Fig. 2B). CCL5 RANTES In chemotaxis assays using a wider range of chemokine concen-

CCR2 CCL2 MCP-1 trations, CCL22/MDC recruited NKL cells with an EC50 of 5 pM, CCR3 CCL11 Eotaxin ␤ ␣ CCL19/MIP-3 exhibited an EC50 of 3 nM, and CXCL12/SDF-1 CCR4 CCL22 MDC C CCL17 TARC exhibited an EC50 of 4 pM (Fig. 2 ). In addition, a ligand specific CCR5 CCL4 MIP-1␤ for CX3CR1 (the chemokine domain of CX3CL1/fractalkine, ␣ CCR6 CCL20 MIP-3 herein termed “FK-CK”) recruited NKL cells with an EC50 of 50 CCR7 CCL19 MIP-3␤ nM. Ligands for CCR8, CCR9, CCR10, CXCR3, and CXCR6 CCL21 SLC failed to recruit the NKL cells (Fig. 2C). CCR8 CCL1 I-309 CCR9 CCL25 TECK The KHYG-1 cell line was analyzed in a similar fashion. By CCR10 CCL27 CTACK flow cytometry, the cells were positive only for CCR6, CXCR3, CXCR1,2 CXCL8 IL-8 and—to a very low extent—CXCR4 (Fig. 3A). In chemotaxis as- CXCR2 CXCL2 Gro␤ says using three concentrations of ligand, only CCL20/MIP-3␣, CXCR3 CXCL11 I-TAC ␣ CXCL10 IP10 CXCL11/I-TAC, and CXCL12/SDF-1 induced migration of the CXCR4 CXCL12 SDF-1␣ KHYG-1 cells (Fig. 3B). In chemotaxis assays using a broader CXCR5 CXCL13 BLC range of ligand concentrations, CCL20/MIP-3␣ recruited the cells CXCR6 CXCL16 CXCL16 with an EC50 of 120 pM, CXCL11/I-TAC exhibited an EC50 of CX3CR1 CX3CL1 Fractalkine ␣ 110 pM, and CXCL12/SDF-1 exhibited an EC50 of2nM The Journal of Immunology 7835

FIGURE 2. Chemokine receptor expression on the NKL cell line. A, Flow cytometric analysis. NKL cells were stained with Abs specific for human chemokine receptors (filled histograms) vs isotype-matched control Abs (open histograms). B, Chemotaxis assay. NKL cells were tested for migration to chemokines at three concentrations (1, 10, and 100 nM), and then the cells in the bottom chamber were quantified by DNA content. Data were plotted as the ratio of migration induced by chemokine to the migration induced by buffer. C, Full-dose chemotaxis assay. NKL cells were tested for migration to chemokines at multiple doses, ranging from 3 pM to 1 ␮M, and then the migrated cells were quantified by DNA content. Data were plotted as the extent of migration (in arbitrary units of fluorescence) vs chemokine concentration, and curve-fitting was performed by nonlinear regression.

(Fig. 3c); ligands for CCR3 (CCL11/eotaxin), CCR7 (CCL21/SLC), ligand, recruited the cells with an EC50 of 410 pM (Fig. 4C). Table and CX3CR1 (FK-CK) failed to recruit the KHYG-1 cells (Fig. 3C). II summarizes the data obtained from the flow cytometric and che- The NK-92 cell line was positive only for CXCR3, although a motaxis assays for these three NK cell lines. very slight staining with the CCR1 and CXCR4 Abs was usually observed (Fig. 4A). In chemotaxis assays using three concentra- Determination of chemokine receptor expression on primary tions of ligand, ligands for CXCR3 (CXCL11/I-TAC) and CXCR4 NK cells (CXCL12/SDF-1␣) induced migration of the cells; in addition, Because the published studies are discrepant with regard to che- migration of the cells toward ligands for CCR1 and CCR5 (CCL5/ mokine receptor expression on primary peripheral blood NK cells, RANTES) and CCR7 (CCL19/MIP-3␤) was observed, but only at we sought to determine which chemokine receptors are expressed the highest concentrations (Fig. 4B). In full-dose chemotaxis as- by these cells. Toward this end, we enriched freshly isolated hu- says, CXCL11/I-TAC recruited the NK-92 cells with an EC50 of 6 man NK cells from the peripheral blood of two healthy human ␣ pM and CXCL12/SDF-1 exhibited an EC50 of 3 nM, whereas donors and performed chemotaxis assays in vitro. Enriched NK cells ␤ ϩ Ϫ CCL19/MIP-3 recruited a small number of cells with an EC50 of (95% CD56 CD3 ) were exposed to increasing concentrations of 12 nM (Fig. 4C). Ligands for CCR4 (CCL22/MDC), CCR5 ligands for CCR4 (CCL22/MDC), CCR5 (CCL4/MIP-1␤), CCR6 (CCL4/MIP-1␤), and CCR10 (CCL27/CTACK) failed to induce (CCL20/MIP-3␣), CCR7 (CCL21/SLC), CXCR1,2 (CXCL8/IL-8), migration, whereas the ligand for CX3CR1 recruited a small num- CXCR3 (CXCL11/I-TAC), and CXCR4 (CXCL12/SDF-1␣) (Fig. 5). ber of cells at the two highest doses (Fig. 4C). CCL3/MIP-1␣,a The ␣ chemokines CXCL8/IL-8, CXCL11/I-TAC, and CXCL12/ CCR1 ligand, did not induce migration of the NK-92 cells; how- SDF-1␣ induced migration of both donors’ cells: CXCL8/IL-8 ex- ␤ ever, N-terminally truncated CCL23/CK 8, a more potent CCR1 hibited EC50s of 250 and 70 pM for donor 1 and donor 2, respectively, 7836 CHEMOKINE RECEPTORS ON NK CELLS

FIGURE 3. Chemokine receptor expression on the KHYG-1 cell line. A, Flow cytometric analysis. KHYG-1 cells were stained with Abs specific for human chemokine receptors (filled histograms) vs isotype-matched control Abs (open histograms). B, Chemotaxis assay. KHYG-1 cells were tested for migration to chemokines at three concentrations (1, 10, and 100 nM), and then the cells in the bottom chamber were quantified by DNA content. Data were plotted as the ratio of migration induced by chemokine to the migration induced by buffer. C, Full-dose chemotaxis assay. KHYG-1 cells were tested for migration to chemokines at multiple doses, ranging from 3 pM to 1 ␮M, and then the migrated cells were quantified by DNA content. Data were plotted as the extent of migration (in arbitrary units of fluorescence) vs chemokine concentration, and curve-fitting was performed by nonlinear regression.

whereas CXCL11/I-TAC exhibited EC50s of 1.6 and 0.8 nM and for a number of chemokine receptors in combination with Abs ␣ ␤ CXCL12/SDF-1 exhibited EC50s of 7.1 and 3.5 nM. None of the specific for CD3, CD14, CD16, CD19, and CD56. For both chemokines induced migration. A chemotaxis assay was also per- CD56bright and CD56dim NK populations, data were plotted in che- formed on pooled IL-2-activated NK cells from two more donors; mokine receptor vs CD16 dot plots to reveal potentially rare sub- migration was induced by CXCL8/IL-8, CXCL11/I-TAC, and sets (data from a representative donor are depicted in Fig. 6). CXCL12/SDF-1␣ but not by CCL22/MDC, CCL20/MIP-3␣,orli- CCR1, CCR4, CCR5, CCR6, CCR7, and CXCR6 were detected gands for CCR9 (CCL25/TECK), CXCR6 (CXCL16), or CK3CR1 on some but not all NK cells in each of the 11 donors, with all but (FK-CK) (data not shown). CCR4 preferentially expressed in the CD56bright population (Table To determine whether chemokine receptors are expressed on III). The proinflammatory chemokine receptors CCR1 and CCR5 minor subpopulations of primary NK cells, flow cytometric anal- were detected on approximately one-third of CD56bright cells, al- ysis was performed on PBMC from 11 donors, using Abs specific though that amount is likely an underestimate due to the exclusion The Journal of Immunology 7837

FIGURE 4. Chemokine receptor expression on the NK-92 cell line. A, Flow cytometric analysis. NK-92 cells were stained with Abs specific for human chemokine receptors (filled histograms) vs isotype-matched control Abs (open histograms). B, Chemotaxis assay. NK-92 cells were tested for migration to chemokines at three concentrations (1, 10, and 100 nM), and then the cells in the bottom chamber were quantified by DNA content. Data were plotted as the ratio of migration induced by chemokine to the migration induced by buffer. C, Full-dose chemotaxis assay. NK-92 cells were tested for migration to chemokines at multiple doses, ranging from 3 pM to 1 ␮M, and then the migrated cells were quantified by DNA content. Data were plotted as the extent of migration (in arbitrary units of fluorescence) vs chemokine concentration, and curve-fitting was performed by nonlinear regression.

of CCR1dim and CCR5dim cells during quantitation. CCR1 and Within the positive donors, CCR9 and CXCR5 were detected on CCR5 were also detected on ϳ10% of CD56dim NK cells. CCR7, 2–3% of CD56bright NK cells and on only 0.2–0.8% of CD56dim a receptor critical for entry of leukocytes into secondary lymphoid NK cells. organs, was detected on at least 60% of CD56bright cells, but Ͻ5% of CD56dim NK cells. CCR4, CCR6, and CXCR6, chemokine re- ceptors important for targeting leukocytes to specific tissues, were Discussion detected on ϳ4, 9, and 24% (respectively) of CD56bright NK cells In this study, we have determined that peripheral blood NK cells and on ϳ7, 3, and 4% of CD56dim NK cells. are heterogeneous in their expression of certain chemokine recep- In contrast, CCR9 and CXCR5 were detected on NK cells from tors, and thereby have the capacity for differential trafficking only a few donors (Table III). CCR9, a receptor important for within the body. Using flow cytometry, we identified subsets of homing of leukocytes to the intestines and possibly lung, was de- peripheral blood NK cells expressing the chemokine receptors tected on a small subset of both CD56bright and CD56dim NK cells CCR1, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR5, and CXCR6. from one donor (the one depicted in Fig. 6) and on a small subset (At present, we do not know whether these chemokine receptors of CD56dim NK cells from three other donors (data not shown). are on the same NK cell or are on different NK cells.) The che- CXCR5, a receptor important for homing of leukocytes to lym- mokine receptors were detected on both CD56bright and CD56dim phoid follicles, was detected on a small subset of both CD56bright NK cells, though each receptor, except CCR4, was expressed pref- and CD56dim NK cells from two donors (one depicted in Fig. 7) erentially on the CD56bright NK population. CCR1, CCR4, CCR5, and on CD56dim NK cells from a third donor (data not shown). CCR6, and CXCR6 were detected on all 11 donors analyzed, 7838 CHEMOKINE RECEPTORS ON NK CELLS

Table II. Analysis of chemokine receptor expression on NK cell lines by migration assay and FACS

NKL Migration FACS KHYG-1 Migration FACS NK-92 Migration Flow Cytometry

CD56 Ϫ/dim ϩ Bright CD16 ϩϪ Ϫ ϪϪ ϪϪϩ␤ ϩ CCR1 (CK 8; EC50 410 pM) CCR2 ϪϪ ϪϪ Ϫ Ϫ CCR3 ϪϪ ϪND ϪϪ ϩ ϩϪϪ Ϫ Ϫ CCR4 (MDC; EC50 5 pM) CCR5 ϪϪ ϪϪ Ϫ Ϫ ϪϪϩ␣ ϩϪ Ϫ CCR6 (MIP-3 ;EC50 120 pM) ϩ ␤ ϩϪϪϩϪ ␤ Ϫ CCR7 (MIP-3 ;EC50 3 nM) / (MIP-3 ;EC50 12 nM) CCR8 ϪϪ ϪϪ Ϫ Ϫ CCR9 ϪϪ ϪϪ Ϫ Ϫ CCR10 Ϫ ND Ϫ ND Ϫ ND CXCR1 ϪϪ ϪϪ Ϫ Ϫ CXCR2 ϪϪ ϪϪ Ϫ Ϫ Ϫϩϩ ϩϩ ϩ CXCR3 (I-TAC; EC50 110 pM) (I-TAC; EC50 6 pM) ϩ ␣ ϩϩ ␣ ϩϩ␣ ϩ CXCR4 (SDF-1 ;EC50 4 pM) (SDF-1 ;EC50 2 nM) (SDF-1 ;EC50 3 nM) CXCR5 ϪϪ ϪϪ Ϫ Ϫ CXCR6 ϪϪ ϪϪ Ϫ Ϫ ϩ Ϫ ϩ Ϫ Ͼ CX3CR1 (FK-CK; EC50 50 nM) ND ND / (FK-CK; EC50 150 nM) ND whereas CCR9 and CXCR5 were detected on a subset of the do- the other chemokine receptors were present on very minor subsets nors. The size of the NK subsets expressing chemokine receptor of the CD56dim NK cell population or were expressed on the rare varied from Ͻ1% (e.g., CCR9 and CXCR5 on CD56dim NK cells) CD56right NK cell population, we were unable to detect migration to the vast majority (e.g., CCR7 on CD56bright NK cells). of these cells in chemotaxis assays, which are not sufficiently sen- In addition, we have described the complete chemokine receptor sitive to detect the migration of very rare cell types within a het- profiles of three commonly studied NK cell lines: NKL (10), erogeneous population. We attempted to measure chemokine re- KHYG-1 (11), and NK-92 (12). The cell lines each expressed ceptor signaling in enriched primary NK cell preparations, using a CXCR3 and CXCR4, but varied in expression of other chemokine calcium mobilization assay, but never detected robust signaling receptors. NKL, which was CD56Ϫ/dimCD16ϩ, expressed CCR4, with any chemokine. CCR7, and CX3CR1. KHYG-1 was CD56ϩCD16Ϫ and expressed Our data obtained using primary NK cells have a few discrep- CCR6, whereas NK-92 was CD56brightCD16Ϫ and expressed CCR1. ancies with the two prior surveys of chemokine receptor expres- The expression of CCR7 on NKL but not KHYG-1 appears sion by human NK cells, and the two prior studies were themselves contradictory, because we detected CCR7 on a much higher very discrepant. Campbell et al. (8) performed flow cytometric and frequency of CD56brightCD16Ϫ primary NK cells than chemotaxis assays on freshly isolated CD56dimCD16ϩ and CD56bright CD56dimCD16ϩ cells. However, the primary tumor from which CD16Ϫ cells separately; the former cells were found to be positive KHYG-1 developed was originally described as CD16ϩ, with only for CXCR1–4 and CX3CR1, whereas the CD56brightCD16Ϫ CD16 disappearing during culture (11). In addition, we did detect subset was found to be positive only for CCR5, CCR7, CXCR3, CCR7 on a small percentage of CD56dimCD16ϩ primary NK cells; CXCR4, and, to a small extent, CX3CR1. Unlike in our study, Camp- the NKL line might have been derived from one of these rare cells. bell et al. (8) did not detect CCR4, CCR6, CCR9, CXCR5, or CXCR6 Results from chemotaxis assays correlated well with flow cyto- on NK cells. Inngjerdingen et al. (9) performed both assays on total metric data, with the exception that CXCR3 on NKL was non- freshly isolated NK cells, with no distinction between the functional and CCR1 on NK-92 was functional only for CCL5/ CD56dimCD16ϩ and CD56brightCD16Ϫ subpopulations. By flow cy- RANTES and CCL23/CK␤8⌬24, but not CCL3/MIP-1␣. The tometric analysis, these NK cells were found to be positive for presence of CCR1, CCR4, CCR6, and CCR7 variably on NK cell CXCR4, CCR7, and, to a lesser extent, CXCR3 and CCR4. By che- lines supports the primary NK cell flow cytometric data, indicating motaxis assays, the freshly isolated NK cells migrated to ligands for that these receptors are expressed variably on primary NK cells. CCR2, CCR4, CCR6, CCR7, CXCR3, and CXCR4; CCR9 and Enriched primary NK cells migrated to ligands for CXCR1, CXCR6 were not evaluated. The reasons for the discrepancies in che- CXCR3, and CXCR4, as reported previously (8). Because many of mokine receptor detection between our study, the Campbell et al. (8)

FIGURE 5. Chemotactic analysis of fresh NK cells. Freshly isolated NK cells from two donors were tested for migration to chemokines at multiple doses, ranging from 3 pM to 1 ␮M, and then the migrated cells were quantified by DNA content. Data were plotted as the extent of migration (in arbitrary units of fluorescence) vs chemokine concentration, and curve-fitting was performed by nonlinear regression. The Journal of Immunology 7839

FIGURE 6. Chemokine receptor expression on NK cells in freshly prepared human PBMC. Lineage (CD3, CD14, CD19)-negative cells (histogram, top left) were analyzed for CD56 vs CD16 expression (dot plot, top right). CD56bright cells and CD56dim cells were gated and each analyzed for chemokine receptor vs CD16. CD56bright cells were observed to include CD16Ϫ and CD16dim cells. Chemokine receptor-positive cells were quantitated by quadrant analysis (CD56bright NK cells) or by gating (CD56dim NK cells).

study, and the Inngjerdingen et al. (9) study are not known, but may peripheral blood NK cells, primarily the CD56bright cells, may vary relate to differences in cell isolation techniques and/or the Abs used in their abilities to migrate to different tissues. CCR1 and CCR5 for detection of the chemokine receptors. Importantly, both of the are chemokine receptors that have been implicated in response to prior studies analyzed histograms displaying the flow cytometric data inflammation, suggesting that subsets of CD56bright and CD56dim to evaluate receptor expression; however, histograms are not sensitive NK cells have the capacity to migrate to sites of inflammation. for the detection of very minor subpopulations. Instead, our analysis CCR7 is critical for entry of leukocytes into secondary lymphoid used two-parameter dot plot displays of the flow cytometry data, in organs, such as lymph nodes; our results support the hypothesis which every cell analyzed is visualized—a more sensitive technique that CCR7-positive CD56bright NK cells are immunoregulatory for detecting and displaying very rare cell populations. cells with roles inside secondary lymphoid organs, whereas CCR7- Our observation that NK cell subsets express CCR1, CCR4, negative CD56dim NK cells are cytotoxic effectors maintained in CCR5, CCR6, CCR7, CCR9, CXCR5, and CXCR6 suggests that the peripheral blood. CCR4 is expressed on cutaneous T cells and is thought to be necessary for homing to skin, whereas CCR9 is expressed on mucosal T cells and is thought to be necessary for Table III. Chemokine receptor expression in primary NK cell homing to the intestine and perhaps lung (13). CCR6 has been populations implicated in homing of leukocytes to skin (i.e., Langerhans cells), intestine (14), and lung (15); CXCR6 has been implicated in hom- No. of Donorsa Averageb SD ing of T cells to rheumatoid arthritic joints (16) and liver (17), and CCR1 CXCR5 is critical for migration of T cells into follicles in CD56bright 9/9 33.8 19.0 dim lymphoid organs (18) and lymphoid aggregates in nonlymphoid CD56 9/9 11.6 8.9 organs (19–21). It remains to be seen whether NK cells expressing CCR4 CD56bright 11/11 3.6 2.0 these chemokine receptors are enriched at the receptors’ target CD56dim 11/11 6.8 5.0 tissues. However, the presence of these chemokine receptors on CCR5 minor subsets of NK cells in peripheral blood may provide the bright CD56 9/9 31.3 22.0 mechanism whereby subsets of NK cells migrate to these tissues CD56dim 9/9 10.4 9.0 CCR6 during immune responses to infectious agents and cancer. CD56bright 11/11 9.3 3.8 CD56dim 11/11 2.6 2.8 CCR7 CD56bright 11/11 59.7 20.0 CD56dim 11/11 3.3 2.4 CCR9 CD56bright 1/11 2.1 CD56dim 4/11 0.4 0.1 CXCR5 CD56bright 2/11 2.7 0.1 CD56dim 3/11 0.5 0.2 CXCR6 CD56bright 11/11 24.0 18.0 CD56dim 11/11 3.7 3.4 FIGURE 7. CXCR5 expression on NK cells in freshly prepared human bright dim a PBMC. CD56 and CD56 NK cells, identified as indicated in the Number of donors expressing the chemokine receptor/number of donors ϩ analyzed. Fig. 5 legend, were analyzed for CXCR5 vs CD16 expression. CXCR5 b Percentage of cell population expressing the receptor. cells were quantitated by quadrant analysis. 7840 CHEMOKINE RECEPTORS ON NK CELLS

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