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The Journal of Immunology, 2006, 177: 3669-3676.
Copyright © 2006 by The American Association of Immunologists, Inc.

CD4 Expression on Activated NK Cells: Ligation of CD4 Induces Cytokine Expression and Cell Migration1

Helene B. Bernstein2,*, Mary C. Plasterer*, Sherrie E. Schiff*, Christina M. R. Kitchen§, Scott Kitchen{dagger} and Jerome A. Zack{dagger},{ddagger}

* Department of Obstetrics and Gynecology, {dagger} Department of Medicine, and {ddagger} Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, and § Department of Biostatistics, School of Public Health, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells, we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity, and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines {gamma}-IFN and TNF-{alpha} than are CD4– NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16, providing another function for the CD4 molecule on NK cells. Thus, the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production, in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are an important component of the innate immune system, comprising 10–15% of PBMC (1). In contrast to cells of the adaptive immune system, NK cells can quickly lyse target cells without prior sensitization. NK cells lack the Ag-specific receptors found on B and T lymphocytes but interact with target cells via killer Ig-like receptors (KIRs)3 and C-type lectin-like receptors. KIRs may have long or short cytoplasmic tails (L or S forms, respectively). The L form KIR has two or more tyrosine-based inhibitory motifs within its cytoplasmic domain and sends a transient inhibitory signal to the NK cell upon binding its cognate ligand (2, 3). The S forms of KIR lack these domains and send an activating signal following ligand binding.

Similar to the KIRs, the C-type lectin receptors also have both inhibitory and activating forms. The ligands for NK cell inhibitory receptors are often MHC class I (MHC I) molecules (3, 4, 5). Therefore, MHC I-positive target cells bind inhibitory receptors on the NK cell surface, and target cells lacking MHC I activate NK cells by failing to provide the appropriate ligand to the inhibitory receptor. Several activating receptors, including NKG2C, NKG2D, LFA-1, DNAM, and 2B4 and the natural cytotoxicity receptors, NKp30, NKp44, and NKp46, also exist on NK cells, and ligation of these receptors can also lead to activation and increased cytotoxicity (5). NK cell responses are dependent on the specific receptor bound, receptor ligand affinity and the number of ligands present on the target cells. Binding of cell surface receptors on NK cells may lead to inhibition, cellular activation, cytokine production, enhanced cytotoxicity, or even apoptosis (5, 6, 7).

Recently, we have isolated NK cells from tissue that express the CD4 molecule on their surface. CD4 is normally expressed on helper T cells, where it plays an important role in the recognition of MHC molecules on the surface of adjacent cells. However, it can also be expressed on many other hemopoietic cell types, including macrophages, eosinophils, neutrophils, and CD8+ T cells (8, 9, 10, 11, 12, 13).

The CD4 molecule is a 55-kDa monomeric membrane glycoprotein containing four tightly packed, Ig-like domains (D1-D4) (14). Domains D1 and D2 are distal to the cell surface and are separated by a hinge domain from the D3 and D4 domains, located proximal to the cell surface. HIV binds to the D1 region of CD4 (15), and the binding site for MHC class II (MHC II) includes sequences that lie on the lateral face of D1 and may include some D2 residues (16). CD4 binds to the beta2 region of MHC II, a conserved region of the molecule distinct from the TCR-MHC binding site. Therefore, CD4 can bind to MHC at the same time that TCR interacts with the MHC peptide, forming a ternary complex with synergistic abilities to provide signals during Ag binding.

In addition to acting as a coreceptor for TCR-MHC peptide interactions, CD4 also functions as the receptor for the chemotactic factor IL-16, a proinflammatory cytokine that binds to the D4 region of CD4 (17, 18). IL-16 is produced by activated CD4+ and CD8+ T cells, B cells, and bronchial epithelial cells as a precursor molecule, which is processed and secreted as a 17-kDa protein. Binding of the biologically active IL-16 tetramer to CD4 on T cells results in an increase in intracellular calcium and inositol triphosphate levels and autophosphorylation of p56lck (19). This signaling induces chemotaxis of the CD4+ T cell along a concentration gradient of IL-16 and can be inhibited by monomeric Fabs of the OKT4 mAb, which is specific for the D4 region but not by Abs to other epitopes of CD4.

Although the function of CD4 on CD4+ T cells has been explored in great detail, much less is known about the role of CD4 on other hemopoietic cell types. We and others (9, 11, 13) have shown that activation of CD8+ T cells from peripheral blood induces the expression of CD4 on the cell surface. These CD4dim/CD8+ T cells can be infected with HIV, and CD4 appears to play a functional role on these cells. Ligation of CD4 on CD4dim/CD8+ T cells causes intracellular signaling, via Src family kinases, and has been shown to induce {gamma}-IFN expression and Fas ligand expression (12, 20). Additionally, these CD4dim/CD8+ T cells migrate in response to an IL-16 gradient, and migration can be inhibited with PP1, an inhibitor of Src-family kinases (Lck) (12). Thus, the CD4 molecule on the surface of NK cells could function in a similar manner to direct these cells to sites of IL-16 expression and cause NK cells to be susceptible to HIV infection. In addition, because NK cells are functionally distinct from T lymphocytes, it is possible that CD4 could perform other functions on NK cells.

In the current studies, we found low levels of CD4 expression on peripheral blood NK cells; this finding is consistent with other researchers in that <3% of peripheral blood NK cells express the CD4 molecule on their surface (21). However, we found high levels of CD4 expression on NK cells derived from tissues. This led us to undertake studies to explore the function of the CD4 molecule on NK cells using a culture system that allows robust CD4 expression on primary NK cells in vitro. Through these studies, we determined that CD4 has a role in NK cell function in increasing cytokine production and directing cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
NK cell purification

Human subjects research approval was obtained from University of California, and informed consent was obtained from volunteers as appropriate. Mononuclear cells from whole blood or buffy coats were purified over Ficoll-Hypaque density gradients. Thymic and tonsillar tissue was mechanically dispersed and passed though a cell strainer to obtain a single-cell suspension, and mononuclear cells were purified as described above. NK cells were obtained by negative selection using column-based immunomagnetic cell separation techniques (StemCell Technologies) as described previously (22). Following purification, NK cell purity was confirmed to be >99% via flow cytometry as described below. Approximately 80% of the purified NK cells expressed CD56, and these cells were negative for the lineage markers CD3, CD19, and CD14 and were also negative for TCR {alpha}beta and {gamma}{delta}, as assessed by labeled Ab staining, followed by flow cytometric analysis.

NK cell culture

Purified NK cells were placed in culture in 96-well round-bottom plates at a concentration of 5 x 104 cells/ml in medium containing RPMI 1640 supplemented with 10% heat-inactivated FCS, 1 mM L-glutamine, 100 U/ml penicillin, 50 µg/ml streptomycin, 200 U/ml IL-2 (obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health, contributed by Dr. M. Gatley (Hoffman-LaRoche, Nutley, NJ)), 1 ng/ml IL-12 (R&D Systems), and 0.25% PHA (Invitrogen Life Technologies). Optimal IL-12 concentration was determined based on its effect on CD4 expression when up to 10 ng/ml was added to NK cells in culture. A total of 2 x 105 allogeneic, lethally irradiated PBMC and 1.5 x 104 lethally irradiated 721.221G cells were also added to each well (23). After becoming confluent, cells were split or fed every 2 days.

Flow cytometry

NK cell purity was assessed immediately following purification via flow cytometry using the following fluorochrome-labeled Abs: CD56-PE, CD19-FITC, CD14-FITC, TCR {alpha}beta-FITC, TCR {gamma}{delta}-FITC, CD3-ECD, and CD4-allophycocyanin (Beckman Coulter). Analysis was performed on a BD Biosciences FACSCalibur machine using CellQuest software. Cells were gated based on forward and side scatter, quadrant markers were set based on isotype controls, and samples were compensated electronically for overlap in fluorescent emission.

NK cell phenotype was assessed by incubating cells with the appropriate fluorescently labeled mAbs, including CD56-PE, CD8-FITC, CD3-ECD, CD4-allophycocyanin, CD19-FITC, CD94-PE, NKG2D-PE, NKp30-PE, NKp46-PE, NKp44-PE, KAR p50.3-PE, KIR p70-PE (Beckman Coulter), CD56-FITC (eBioscience), and CD69-PE and HLA-DR-PE (BD Biosciences). Cells were then washed with PBS and incubated with 2 µg/ml 7-aminoactinomycin D (7AAD) for 20 min at 4°C. Cells were again washed with PBS and resuspended in 20 µg/ml actinomycin (Sigma-Aldrich) and 1% formaldehyde in PBS to fix the cells. If a FL3-labeled Ab was used, then 7AAD staining was omitted. Flow cytometric analysis was performed on a Becton Dickinson FACSCalibur machine using CellQuest software. Cells were gated based on forward and side scatter in addition to 7AAD exclusion, quadrant markers were set based on the isotype controls, and samples were compensated electronically for overlap in fluorescent emission.

Detection of cytokine production by intracellular Ab staining

Before intracellular Ab staining, cells were incubated with 10 µg/ml brefeldin A (Sigma-Aldrich) for 2–10 h when appropriate. Cells were then incubated with CD56-FITC or CD56-PE and CD4-allophycocyanin for 20 min at 4°C. The cells were then washed and permeabilized using the BD Pharmingen Cytofix/Cytoperm Plus kit per the manufacturer’s instructions, followed by incubation with 20 µl of normal mouse IgG for 15 min to block nonspecific Ab staining (Caltag Laboratories). Abs recognizing one of the following Ags were added to cells: TNF-{alpha} or {gamma}-IFN conjugated to PE (BD Pharmingen), or granzyme A or perforin conjugated to FITC (BD Pharmingen), and incubated for 30 min at 4°C, followed by washing and flow cytometric analysis.

CD4 ligation

The mAb OKT4 was added to NK cells at a ratio of 10 µg/106 cells and incubated in the cold for 30 min. Cells were then washed and resuspended into complete medium containing 8.75 µg goat anti-mouse Ig for 5 min at 37°C. Cells were then rewashed and resuspended in complete medium. At varying times postligation aliquots of cells were removed for intracellular staining and flow cytometric analysis. Negative controls included omission of OKT4 during ligation or the addition of anti-CD56 Ab (in lieu of OKT4) on identical aliquots of cells.

NK cell cytotoxicity assay

Daudi and Ramos cells were obtained from the American Tissue Culture Collection and maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 1 mM L-glutamine, 100 U/ml penicillin, and 50 µg/ml streptomycin. Before use in cytotoxicity assays ~2 x 106 target cells were incubated with 100 µCi of 51Cr (PerkinElmer) for 1.5 h at 37°C, washed, and incubated again for 30 min in RPMI 1640/10% FCS for labeling. NK cells (effectors) were ficolled to remove dead cells; a comparison of equal numbers of viable untouched and ficolled cells indicated that no effector cells were lost during Ficoll purification.

In some experiments, aliquots of NK cells were depleted of CD4+ NK cells using CD4 EasyStep immunomagnetic purification medium per the manufacturer’s recommendations (StemCell Technologies). Complete CD4 depletion of NK cell cultures was verified by labeled Ab staining followed flow cytometric analysis; NK cells used in cytotoxicity assays were >99% CD56+, and no lineage positive (CD3, CD19, CD14) cells were detected. A starting NK cell to target ratio of 20:1 (100,000 cells/well) or 10:1 was used, with 2-fold serial dilutions and three replicates per concentration. Target and effector cells were placed in a 96-well round-bottom plate for 4 h at 37°C. Target cells were also incubated with 0.5% Triton X-100 to measure total release and with culture medium alone to measure spontaneous release, with six replicates each. Supernatants were harvested, placed into a scintillant-coated Luma Plate 96 (PerkinElmer), and read in a Wallac Microbeta Trilux counter (PerkinElmer) when dry. The percent lysis of each type of target cell was calculated by subtracting the spontaneous release from the experimental counts and dividing it by the total release minus the spontaneous release (means of all replicates were used).

NK cell migration assay

NK cells were placed in the upper well of a Costar 24-well Transwell plate with 8-µm pores. Six hundred microliters of either RPMI 1640/10% FCS, RPMI 1640/10% FCS with 20 ng/ml stromal-derived factor 1{alpha} (SDF-1{alpha}) (R&D Systems), or RPMI 1640/10% FCS with 200 ng/ml IL-16 (R&D Systems) was placed in the bottom well in replicates of three. After 4 h of incubation at 37°C, the cells in each lower well were counted twice, and migration in response to plain medium was set as 100%. Migration in response to chemoattractants was calculated based on the percentage of above spontaneous migration. In some wells, CD4 or CD56 was blocked by adding an excess (10 µg) of the mAb to the NK cells before the migration assay. Cells were incubated with Ab for 30 min at 4°C, washed, and replaced in RPMI 1640/10% FCS before being placed in the migration assay.

Quantitation of TCR-rearrangement excision circles (TREC)

DNA was extracted from NK cells or CD4+ T cells using phenol/chloroform and real-time PCR to quantitate TREC normalized to beta-globin (24) was performed on samples using the oligos, probes, and conditions described by Douek et al. (25). Results are reported as TREC/100,000 cells.

Quantitation of CD4 mRNA

Total RNA from NK cells and CD4-depleted NK cells was extracted using the Qiagen RNeasy kit and protocol. Reverse transcription and quantitative real-time PCR were performed using Qiagen’s SYBR green RT-PCR kit starting with 30 ng of total RNA per reaction. A total of 500 nM gene-specific forward and reverse primers was used as follows: CD4, forward 5'-GCA GTG GCG AGC TGT GGT-3', reverse 5'-GGG TCC CCA CAC CTC ACA GG-3'; and GAPDH, forward 5'-GAA GGT GAA GGT CGG AGT C-3', reverse 5'-GAA GAT GGT GAT GGG ATT TC-3'. The reaction conditions were 30 min at 50°C (one cycle) and 15 min at 95°C (one cycle) for reverse transcription and 15 s at 94°C, 30 s at 60°C, and 30 s at 72°C (40 cycles) for PCR (8, 26). Gene-specific products were continuously measured by means of an iCycler detection system (Bio-Rad). Samples were normalized based on expression of the housekeeping gene GAPDH.

Statistical analysis

Cytokine expression by NK cells was analyzed using the permutation test. Changes in cytokine production following CD4 ligation was assessed via a one-sided paired t test, comparing ligated and mock-ligated samples. NK cell migration was analyzed using the Wilcoxon rank-sum test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tissue derived NK cells express the CD4 molecule in vivo

The CD4 molecule is found on CD4+ T cells in addition to monocytes and macrophages. Recently, this molecule has also been found on activated CD8+ T cells, B cells, eosinophils, and neutrophils (8, 9, 11). Given the recent finding of the CD4 molecule on "atypical" cell types, we sought to determine whether NK cells in vivo express the CD4 molecule on their surface. We found low levels of CD4 expression on NK cells derived from peripheral blood (see Fig. 2A), which is in agreement with prior published studies (21). This led us to evaluate levels of CD4 expression on NK cells isolated from tissues. Within human thymus and tonsil, we found that NK cells represented ~0.4–3% of isolated mononuclear cells (Fig. 1A).


Figure 2
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  		FIGURE 2. NK cells exhibit robust expression of CD4 following in vitro stimulation/culture. NK cells purified by negative selection from PBMC were stained with fluorescently labeled murine mAbs: CD56-PE, CD4-allophycocyanin, and either CD3-ECD or 7AAD (to select for viable cells), percentage of CD4+ cells are shown in the upper right corner of each dot plot. A, Flow cytometric analysis was performed on day 0 before stimulation and (B) on the indicated days following in vitro stimulation with lethally irradiated allogeneic PBMC, irradiated 721.221G cells, PHA, 200 U/ml IL-2 and 1 ng/ml IL-12. C, NK cells from the same donor stimulated in the absence of IL-12 and PHA demonstrated little CD4 expression. Cells are gated based on size and 7AAD exclusion, quadrants were set based on isotype controls, and samples were compensated electronically for overlap in fluorescent emission. D, Reverse transcription followed by quantitative real-time PCR was performed to quantitate CD4mRNA (normalized to GAPDH) in CD4+ NK cells compared with CD4-depleted NK cells. CD4+ NK cells had a 13-fold increase in CD4 mRNA, as compared with CD4-depleted NK cells, (line 1) CD4– NK cells GAPDH mRNA, (line 2) CD4+ NK cells GAPDH mRNA, (line 3) CD4+ NK cells CD4 mRNA, and (line 4) CD4– NK cells CD4 mRNA.

 

Figure 1
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  		FIGURE 1. NK cells express the CD4 molecule in lymphoid tissues in vivo. Tonsillar and thymic tissues were dispersed mechanically into a single-cell suspension, Ficoll purified, and stained with fluorochrome-labeled mAbs as follows: CD56-PE, CD19-FITC, CD3-ECD, CD4-allophycocyanin, and samples were analyzed by flow cytometry. A, Mononuclear cells obtained from tonsil 1 are shown following Ficoll density centrifugation. NK cells represented 0.4–3% of the mononuclear cell population within these tissues, and quadrant statistics are shown in the upper right corner of each dot plot. B, NK cells from tonsil 1 were enriched by negative selection using immunomagnetic columns as described, stained with mAbs to confirm T and B cell depletion, and analyzed by flow cytometry (22 ). C, Histograms detail the percentage of CD4 expression of NK cell-enriched samples gated on CD3–, CD19–, and CD56+ populations from three separate tonsils. NK cells isolated from thymus were stained with Abs as follows: CD56-PE, CD3-ECD, and CD4-allophycocyanin. Histograms represent thymic samples gated to include the cell population, which is CD3– and CD56+. Tissues from five separate donors are shown. D, NK cells isolated from thymus also express NKG2D, NKp46, CD94, and KAR p50.3, and histograms represent a thymic sample gated to include the cell population, which is CD3– and CD56+. In this figure, gating is based on isotype Ab staining, and samples were compensated electronically for overlap in fluorescent emission.

 
NK cells were enriched from these tissues by negative selection (27, 28), and CD4 expression was assessed by fluorescent-labeled Ab staining followed by flow cytometry and analysis. We found that NK cells (histogram gated to show cells that are CD3–, CD19–, and CD56+) purified from human tonsil expressed CD4 at levels ranging from 7.5 to 13.8% (Fig. 1, B and C). The absence of lineage-specific markers, including CD3 and CD19 and the presence of CD56, NKG2D, NKp46, CD94, and KAR p50.3 (Fig. 1D), on these cells confirms that they are NK cells and not a subset of activated T cells, B cells, or NKT cells. NK cells from human thymus (histogram gated to show cells that are CD3– and CD56+) expressed levels of CD4 ranging from 60 to 80% as shown in Fig. 1C. Thus, CD4+ NK cells exist in lymphoid tissue in vivo at levels substantially higher than those found in peripheral blood.

NK cells cultured in vitro demonstrate robust CD4 expression

While a substantial percentage of tissue-derived NK cells express CD4, it is difficult to obtain sufficient quantities of these cells for functional analysis. To study the function of the CD4 molecule on NK cells, we established an in vitro system that induces CD4 expression on peripheral blood NK cells and permits the generation of large quantities of CD4-expressing NK cells for functional studies (see Materials and Methods). As shown in Fig. 2A, fresh peripheral blood NK cells express very little CD4, and the majority of the cells express CD56. However, after stimulating these NK cells in culture, we were able to induce substantial levels of CD4 expression and in excess of 70 and >99% of cells were CD3–,CD56+ (Fig. 2B). CD4+ NK cells also had a 13-fold increase in CD4 mRNA levels, compared with identically cultured NK cells which did not express the CD4 molecule on their surface, as quantitated by real-time PCR (Fig. 2D).

CD4 expression increases soon after stimulation, peaks at day 8–10, and is stable for at least 3 wk when NK cells are maintained in culture under these conditions. Furthermore, IL-12, a cytokine that is stimulatory to NK cells, causes an increase in CD4 expression in a dose-dependent manner; however, CD4 expression was dependent on both IL-12 and PHA (Fig. 3).


Figure 3
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  		FIGURE 3. NK cells express CD4 following in vitro stimulation/culture. NK cells purified by negative selection were stained with fluorescently labeled murine mAbs: CD56-PE, CD4-allophycocyanin, and CD3-ECD, as well as 7AAD to select for viable cells. Flow cytometric analysis was performed on either day 0 (A) or day 16 (B) after in vitro stimulation with allogeneic irradiated feeders, 721.221 cells, PHA, 200 mU/ml IL-2, and varying concentrations of IL-12. Cells are gated based on size and 7AAD exclusion.

 
NK cells stimulated with IL-2 alone (in the absence of IL-12 and PHA), a standard culture condition for NK cells in vitro, exhibited a small, transient increase in CD4 expression at day 5 but remained by and large CD4 negative (Fig. 2C). Before and after stimulation, cells were analyzed by labeled Ab staining followed by flow cytometric analysis to confirm NK cell purity and rule out the presence of T and NKT cells. CD56+ cells were confirmed to be NK cells by their absence of CD3, CD14, CD19, TCR {alpha}beta, and TCR {gamma}{delta} and NKT cell-associated markers, including V{alpha}14, on their surface and the presence of CD16 and the NK cell cytotoxicity receptors NKp30, NKp40, and NKp46 on their surface, as shown at day 0 and day 6 of culture (Fig. 4). As an additional control documenting a lack of T cell contamination within our cultures, we quantitated TREC using quantitative real-time PCR. Using DNA isolated from NK cells and CD4+ T cells isolated from the same individual and cultured under identical conditions, we found no detectable TREC in our NK cell culture (Table I). TREC were detectable in the CD4+ T cell culture, demonstrating that our assay is capable of detecting TREC in T cells cultured under the same conditions and provides additional confirmation that our CD56+/CD4+ cells are not contaminated by CD4+ T cells and are in fact NK cells.


Figure 4
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  		FIGURE 4. NK cell phenotype following activation. NK cells purified by negative selection from PBMC were stained with fluorescently labeled murine mAbs: CD4-allophycocyanin, NKp30-PE or CD69-PE or HLA-DR-PE or CD16-PE, CD56-FITC, and 7AAD (to select for viable cells). Flow cytometric analysis was performed on day 0 before stimulation and on day 6 following in vitro stimulation. Histograms show cells gated for viability and CD56 expression. Day 6 histograms represent the CD4+ and CD4– NK cell populations.

 

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  		Table I. TREC frequency

 
CD4+ NK cells effectively mediated NK cell cytotoxicity

One of the primary determinants of NK cell function is the ability to lyse susceptible target cells in cytotoxicity assays. We evaluated the lytic activity of CD4+ NK cells in a standard killing assay using 51Cr-labeled target cells. As a positive control, we stimulated purified NK cells from the same donor with 200 U/ml IL-2 and lethally irradiated, allogeneic feeder cells. This is a standard stimulation condition for NK cells in vitro, and <1% of the cells stimulated by this method expressed CD4 by FACS analysis. We found that our CD4+ NK cells were able to effectively lyse Ramos cells, a non-Hodgkin’s B cell lymphoma cell line (Fig. 5), in a standard 4-h cytotoxicity assay. This indicates that our culture conditions do not alter NK cell function and suggests that CD4+ NK cells maintain their cytolytic effector potential.


Figure 5
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  		FIGURE 5. CD4+ NK cells effectively mediate NK cell cytotoxicity. CD4-expressing NK cells stimulated with IL-2, IL-12, PHA, or NK cells from the same donor cultured under conditions that do not induce CD4 expression (IL-2) were incubated with 51Cr-labeled Ramos cells for 4 h using E:T ratios ranging from 0.625 to 20:1. 51Cr release from target cells into cell culture supernatants was quantitated to measure NK cell cytotoxicity. Total release (targets lysed with 0.5% Triton X-100), and spontaneous release (no effector cells) controls were performed with each assay and percentage of cytotoxicity was calculated by comparing samples to the total release (100%) and spontaneous release (0%) controls. Each condition was assayed in triplicate and averages are shown. These data are representative of five independent experiments performed with separate donors.

 
As not all NK cells express CD4 in culture, we sought to determine whether there is a qualitative difference in NK cell cytotoxicity between NK cells that express CD4- and CD4-negative NK cells cultured similarly. As ligand binding on NK cells has been shown to lead to apoptosis, we designed our experiment to purify cells using negative selection, leaving remaining cells untouched by Ab. Following stimulation of NK cells to induce CD4 (Fig. 6A), we depleted a portion of these cultures of CD4+ NK cells using positive immunomagnetic selection. Depletion of CD4+ NK cells was verified by labeled Ab staining followed by flow cytometric analysis (Fig. 6B). Identical numbers of stimulated NK cells (47% CD4+) and CD4-depleted NK cells from identical culture conditions were then tested as effector cells in a 4-h NK cell cytotoxicity assay using radiolabeled Ramos and Daudi cells as targets. There were minimal differences between NK cell killing of CD4+ and CD4– NK cells cultured under identical conditions, indicating that CD4 expression by NK cells does not greatly impact their ability to mediate NK cell cytotoxicity (Fig. 6C).


Figure 6
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  		FIGURE 6. Depletion of CD4+ NK cells does not greatly impact cytolytic activity. A, NK cells were stimulated in culture for 8 days to induce CD4 expression, and aliquots of cells were stained with mAbs specific for CD4-allophycocyanin and CD56-PE followed by flow cytometric analysis. B, Additional aliquots of these cells were depleted of CD4+ NK cells using immunomagnetic separation methods, and CD4 depletion was confirmed by mAb staining and flow cytometric analysis. C, Equal numbers of either untouched or CD4-depleted NK cells from a single donor were incubated with 51Cr-labeled Ramos or Daudi cells for 4 h using E:T ratios ranging from 0.625 to 20:1. 51Cr release from target cells into cell culture supernatants was quantitated to measure NK cell cytotoxicity. Each condition was assayed in triplicate and averages are shown. These data are representative of five independent experiments done with separate donors.

 
CD4+ NK cells produce {gamma}-IFN and TNF-{alpha}

In addition to mediating NK cell killing and Ab dependent cytotoxicity, NK cells produce the proinflammatory cytokines {gamma}-IFN and TNF-{alpha} as part of the innate immune response. To further assess the function of NK cells that express CD4, we quantitated the production of {gamma}-IFN and TNF-{alpha} by CD4+ NK cells via intracellular Ab staining followed by flow cytometric analysis. This methodology enabled us to compare CD4+ and CD4– NK cells from a single culture. We found that a significantly higher percentage of the CD4+ NK cells produced the cytokines {gamma}-IFN and TNF-{alpha} than did CD4– NK cells, p = 0.01 (Fig. 7); although there was not a significant increase in the mean/median fluorescent intensity. These findings suggest that while CD4+ NK cells represent a subset of NK cells with an increase capacity to produce cytokines, others have reported increased cytokine production in CD56bright cells detailing unique functional subsets of NK cells (29).


Figure 7
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  		FIGURE 7. CD4+ NK cells are potent producers of cytokines. Stimulated NK cells were surface stained with CD56-FITC and CD4-allophycocyanin, permeabilized using BD Pharmingen Cytofix/Cytoperm, and stained with mAbs specific for either {gamma}-IFN-PE (A) or TNF-{alpha}-PE (B). The percentage of CD56+/CD4+ and CD56+/CD4– cells producing each cytokine is shown above. This assay was performed on eight different NK cell cultures (different shapes are assigned to each individual), and the average percentage of positive expression is shown with a horizontal line. Differences between cytokine expression in CD4+ and CD4– cells were assessed using the permutation test, a nonparametric test. For {gamma}-IFN and TNF-{alpha}, p = 0.01.

 
Ligation of CD4 on NK cells leads to enhanced cytokine production

After establishing that CD4+ NK cells are more likely to produce the cytokines {gamma}-IFN and TNF-{alpha} than CD4– NK cells, we sought to determine whether CD4 ligation can impact cytokine production by NK cells. Ligation of CD4 was performed by incubating cells with the murine mAb OKT4 followed by a goat anti-mouse Ab to enhance cross-linking and intracellular signaling. NK cells were permeabilized and stained with mAbs specific for {gamma}-IFN and TNF-{alpha} 3 h after ligation. There was a statistically significant increase in the percentage of NK cells producing {gamma}-IFN and TNF-{alpha} following CD4 ligation (Fig. 8). No increase in {gamma}-IFN and TNF-{alpha} production was seen when CD56 was ligated on NK cells, demonstrating that the enhanced cytokine production observed following CD4 ligation is likely specific to the CD4 molecule. These data suggest that interaction of the CD4 molecule on NK cells with an appropriate ligand will lead to enhanced cytokine production, delineating a function for the CD4 molecule on NK cells.


Figure 8
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  		FIGURE 8. Ligation of CD4 on NK cells increases cytokine production. Three hours following CD4 ligation, stimulated NK cells were surface stained with CD56-FITC and CD4-allophycocyanin, permeabilized using BD Pharmingen Cytofix/Cytoperm, and stained with mAbs specific for either {gamma}-IFN-PE or TNF-{alpha}-PE. CD4/CD56-positive cells were gated for analysis. The percentage of CD4+ NK cells from each individual expressing {gamma}-IFN (A) or TNF-{alpha} (B) following ligation or mock ligation was quantitated and is shown in the scatter plot. Statistical analysis was performed using a one-sided paired t test, and p values are shown on each graph. This assay was performed on four different NK cell cultures, and each shape corresponds to one individual.

 
CD4+ NK cells migrate in response to IL-16

IL 16 is a proinflammatory cytokine and chemotactic factor produced by activated CD4+ and CD8+ T cells, B cells, and bronchial epithelial cells (17, 19). IL-16 is also a ligand for CD4 and has been shown to induce the migration of CD4+ cells in vitro (12, 17, 18). To assess the function of the CD4 molecule on NK cells, we performed chemotaxis assays using IL-16 as a chemoattractant. We found that CD4+ NK cells preferentially migrated toward an IL-16 gradient, as compared with spontaneous migration toward "control medium" (Fig. 9A). As an additional control, NK cells from the same donor were stimulated under conditions that do not induce CD4 expression (allogeneic irradiated feeders, irradiated 721.221G cells, and 200 U/ml IL-2). The CD4-negative NK cells did not exhibit migration toward IL-16, suggesting that the chemotaxis of CD4+ NK cells toward IL-16 is specific and dependent on a CD4/IL-16 interaction.


Figure 9
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  		FIGURE 9. CD4+ NK cells migrate toward the cytokine IL-16. A, Purified NK cells were stimulated for 9 days to induce CD4 expression followed by Ficoll gradient centrifugation to remove dead cells. A total of 1 x 106 NK cells (42% CD4+) was placed in the upper chamber of a Costar Transwell plate and allowed to migrate for 4 h in response to 200 ng/ml IL-16 or 20 ng/ml SDF-1{alpha}. Results are the percentage of cells migrating compared with the medium only controls (spontaneous migration, set at 100%). Cell migration was quantified by counting cells in the lower chamber of the chemotaxis chamber at the conclusion of the migration period. Migration of NK cells derived from the same donor, cultured under conditions that did not induce CD4 expression, is shown in the bottom half of A. Triplicate wells were performed for each condition, and averages were calculated. Data shown are representative of the results obtained following three independent experiments using NK cells derived from different donors. For CD4+ NK cells, chemotaxis toward IL-16 was different than medium; p = 0.046 using the Wilcoxon rank-sum test. For NK cells not expressing the CD4 molecule, migration in response to IL-16 was not significantly different than spontaneous migration. B, In additional experiments, aliquots of CD4+ NK cells were pretreated with the Ab OKT4 (to block CD4 on the cell surface) or CD56 before being placed in chemotaxis chambers. OKT4 pretreatment abrogated the ability of CD4+ NK cells to migrate in response to IL-16, but did not impact migration in response to SDF-1{alpha}.

 
To demonstrate that both cell populations were capable of migration, the chemokine SDF-1{alpha} was used as a positive control, and significant migration was observed by both CD4+ and CD4– NK cells. Although the CD4+ NK cells tended to have a greater percentage of change in migration toward the chemokine SDF-1{alpha} (ranging from 157 to 276% in multiple assays) compared with CD4– NK cells (ranging from 151 to 219%), migration was normalized for both cell types based on migration toward medium alone (set to 100%). As there was strong migration by both CD4+ and CD4– NK cells toward SDF-1{alpha}, we do not infer that the difference in migration is physiologically relevant. These results show that CD4+ NK cells are capable of migration and suggest that the CD4 expressed on NK cells functions as a chemotactic receptor.

To confirm that CD4+ NK cell migration in response to IL-16 is mediated by CD4, NK cells were pretreated with anti-OKT4, which blocks the interaction of CD4 and IL-16. Pretreatment of CD4+ NK cells with anti-OKT4 did not impact NK cell migration in response to the chemokine SDF-1{alpha}, which is dependent on CXCR4 expression, demonstrating that Ab binding does not alter NK cell migration nonspecifically. OKT4 pretreatment of CD4+ NK cells completely abrogated migration toward IL-16 in our chemotaxis assay (Fig. 9B), whereas pretreatment with an unrelated Ab (anti-CD56) had no effect on migration. This demonstrates that the CD4 molecule is directly involved in NK cell migration toward the proinflammatory cytokine IL-16.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NK cells facilitate the innate immune response via three primary mechanisms. NK cells can directly kill susceptible target cells, mediate ADCC via their expression of CD16 on the cell surface, and produce cytokines and chemokines that modulate and potentiate the innate immune response. NK cells are characterized by their expression of CD56 and/or CD16 and do not express the lineage markers CD3, CD19, or CD14. The CD8 molecule is expressed on the surface of many NK cells and on all CTL. The CD8 molecule forms a complex with CD3 and the TCR on T cells and is involved in MHC I binding; however, the function of this molecule on NK cells is not well characterized (30, 31). The other major type of T lymphocyte, the helper T cell, expresses CD4 on the surface that interacts with CD3 and the TCR.

Recently, others have identified peripheral blood NK cells that express the CD4 molecule on their surface (21, 32). We find CD4 expression by NK cells to be most prominent on tissue derived NK cells (Fig. 1). As the most significant immune responses in vivo likely occur in lymphoid tissue, where cell-cell contact is enhanced, the high percentage of CD4+ NK cells isolated from these tissues support the concept that CD4+ NK cells are a physiologically relevant subset of NK cells. NK cell frequency in both peripheral blood and lymphoid tissue is less than that of other cell types such as T lymphocytes. As NK cells do not need to have the large repertoire of clonal, genetically rearranged AgRs characteristic of T and B lymphocytes, this could account for their ability to contribute to the host immune response given their lower frequency in vivo.

To better study the function of CD4+ NK cells, we developed a system enabling us to induce robust CD4 expression on NK cells following culture under stimulating conditions (Fig. 2). These cells were confirmed to be NK cells by the presence of CD56 and NK cell cytotoxicity receptors (and the absence of lineage specific markers, including CD3, CD19, and CD14), and CD4 mRNA was detected and quantified by real-time PCR. NK cell expression of CD4 requires IL-12 in a dose-dependent manner, and it is possible that APC could provide this stimulus in vivo. Further studies are being undertaken to evaluate CD4 expression by NK cells following interaction with APC.

As described above, one of the primary functions of NK cells is to mediate killing of susceptible target cells. We have demonstrated that CD4+ NK cells are capable of killing classic NK target cell lines (Figs. 5 and 6). It was of interest that Ramos and Daudi cells express MHC II on their surface, which could potentially interact with CD4. When we depleted CD4+ NK cells from our cultures, we found that removal of CD4+ NK cells does not greatly alter NK cell cytotoxic function (Fig. 6D). This suggests that CD4 expression by NK cells does not impact cytotoxic function. Further studies are needed to determine whether CD4 expression by NK cells impacts their ability to mediate ADCC.

Production of cytokines, including TNF-{alpha} and {gamma}-IFN, is another means by which NK cells mediate the innate immune response. Using intracellular Ab staining followed by flow cytometric analysis, we were able to assess production of TNF-{alpha} and {gamma}-IFN on a single-cell basis. We found that CD4+ NK cells were more likely to produce the cytokines TNF-{alpha} or {gamma}-IFN than were CD4-NK cells from the same donor, cultured under identical conditions (Fig. 7). Others have identified subsets of NK cells with enhanced cytokine production capacity, establishing the presence of unique functional subsets of NK cells (29). Furthermore, following ligation of the CD4 molecule on NK cells, there was an increased number of NK cells producing the cytokines TNF-{alpha} and {gamma}-IFN. This finding provides a mechanism by which CD4-expressing NK cells can modulate the host immune response and establishes a function for the CD4 molecule on NK cells. These cytokines are important mediators of antitumor and antiviral immunity, thus CD4-expressing NK cells could augment the host immune response against viral infections and enhance antitumor immunity.

We and others (12, 19) have shown that CD4+ T cells and CD4dim/CD8+ T cells migrate efficiently toward the chemotactic factor IL-16. We demonstrated that CD4-expressing NK cells were also capable of migrating toward IL-16 in a standard chemotaxis assay, whereas NK cells, which did not express CD4, did not demonstrate chemotaxis toward this cytokine. In addition, by blocking CD4 on NK cells with an excess of mAb against CD4, we were able to completely abrogate migration in response to IL-16, but not SDF-1{alpha}, which binds the CXCR4 molecule, rather than CD4, on NK cells. Furthermore, blocking with an unrelated Ab (anti-CD56) did not impact CD4+ NK cell migration. CD4-mediated migration of NK cells toward IL-16 establishes a second function for the CD4 molecule on NK cells. In vivo, this chemoattractant could allow NK cell migration to sites of acute inflammation, enhancing their ability to augment the innate immune response.

This work has demonstrated that a significant fraction of tissue-derived NK cells express the CD4 molecule on their surface. CD4-expressing NK cells mediate cytotoxicity are potent producers of the cytokines TNF-{alpha} and {gamma}-IFN and represent what we believe to be a highly activated, functionally distinct subset of NK cells. CD4 expression by NK cells also allows them to migrate in response to the proinflammatory cytokine IL-16, which could enhance their ability to contribute to the innate immune response. CD4 expression could also permit HIV infection of NK cells; this possibility is being actively investigated by our group at present. Further studies are also ongoing to assess additional functions of the CD4 molecule on NK cells.


   Acknowledgments
 
We thank Dr. Rebecca Schwiebert for helpful discussion and critical reading of this manuscript, and thank Paul Merriam and Gregory Bristol for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (1 R21 AI58786). H.B.B. is a Building Interdisciplinary Research Careers in Women’s Health Center Scholar (5 K12 HD01400). Back

2 Address correspondence and reprint requests to Dr. Helene B. Bernstein, Department of Obstetrics and Gynecology, David Geffen School of Medicine at University of California, Los Angeles, 10833 Le Conte Avenue, CHS 22-150, Los Angeles, CA 90095-1740. E-mail address: hbernstein{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: KIR, killer Ig-like receptor; MHC I, MHC class I; MHC II, MHC class II; 7AAD, 7-aminoactinomycin D; TREC, TCR-rearrangement excision circle; SDF-1{alpha}, stromal-derived factor 1{alpha}. Back

Received for publication August 4, 2005. Accepted for publication May 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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