FITC-conjugated mouse antihuman CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR1, CXCR3, CXCR4, and CXCR5 or unconjugated monoclonal mouse-antihuman CCR1, CCR2, and CXCR6, as well as PE-conjugated rat antihuman CCR8, PE-conjugated rat antihuman CCR10, and PE-conjugated rat IgG2b, were obtained from R&D Systems Europe Ltd. (Abingdon, UK). FITC-conjugated mouse antihuman CX₃CR1 was purchased from Medical and Biological Laboratories Co. Ltd. (Nagoya, Japan). FITC-conjugated monoclonal mouse antihuman CD3, PE-conjugated monoclonal mouse antihuman CD56, and FITC-conjugated goat anti-mouse were purchased from Becton-Dickinson (San Diego, CA, USA). FITC-conjugated mouse IgG, PE-conjugated mouse IgG, unconjugated mouse IgG, and unconjugated rat IgG were obtained from either Becton-Dickinson or from R&D Systems. Pertussis toxin (PTX), MMF, and DMF were obtained from Sigma-Aldrich (Saint Louis, MO, USA). CCL1, CCL27, CCL28, and CXCL10 were purchased from PeproTech (London, UK).

Buffy coats from normal human volunteers were obtained from the blood bank (Ulleval Hospital, Oslo). Human IL-2-activated NK cells were prepared using Histopaque-1077 (Sigma-Aldrich) and RosetteSep human NK cell enrichment cocktail (Stemcell Technologies, SARL, Grenoble, France). NK cells were negatively selected by removing cells expressing CD3, CD4, CD19, CD36, CD66b, CD123, and glycophorin A. More than 95% of these cells expressed the CD56 molecule but lacked the CD3 molecule as determined by flow cytometric analysis (Figure S1 in Supplementary Material). Purified NK cells were then placed in flasks at 1 × 10⁶/mL and 200 U/mL IL-2 (PeproTech, Rocky Hill, NJ, USA), and then incubated at 37°C in a 5% CO₂ incubator for 5–7 days.

The human myeloid leukemia cell line K562 cells (CCL-243 obtained from American type culture collection “ATCC,” Manassas, VA, USA) or RAJI human lymphoma cells (CCL-214, ATCC), were used as target cells. Target cells were incubated at 1 × 10⁶ cells/mL with 5 μg/mL Calcein AM (Sigma-Aldrich) for 45 min. The cells were pelleted by centrifugation and resuspended in RPMI. To obtain total lysis, these cells were incubated in 96-well plates with 0.05% Triton X, whereas they were incubated with medium alone to obtain total viability. In other cultures, Calcein-AM-labeled cells were incubated at 37°C in a 5% CO₂ incubator with activated NK cells at different NK target cell ratios for 4 h. The plates were centrifuged, supernatants were removed, and replaced with PBS. Fluorescence units (FUs) were measured in Cytofluor plate reader. The percentage of cytotoxicity was calculated according to the following formula: % viability = FU of targets incubated with IL-2-activated NK cells (experimental) minus FU of targets incubated with Triton X, divided by FU of targets incubated in medium only (total viability), minus FU of targets incubated with Triton X (total lysis). In the figures, the 10:1 effector:target (E:T) cell ratio is shown; however, similar results were obtained using other E:T cell ratios (2.5:1 and 5:1). Of note, the media of incubation contains RPMI plus 10% FCS. For anti-CCR10 treatment, the cells were washed and then treated with 10 μg/mL anti-CCR10 or 10 μg/mL isotype control antibody for 45 min at 4°C, washed and examined for viability. Only more than 95% viable cells were then added to Calcein-AM-labeled target cells and incubated for 4 h in the NK cytotoxicity assay.

Nucleopore blind well chemotaxis chambers with a lower well volume of 200 μL were used. A maximum volume of 200 μL medium containing RPMI plus 2% FCS was placed in the lower wells in the presence or absence of various chemokines. Cells (2 × 10⁵) were placed in the upper compartments and incubated for 2 h at 37°C in a 5% CO₂ incubator separated by polycarbonate filters (Nucleopore Polycarbonate 13 mm size 8 UM, Whatman International Ltd., Kent, UK). Filters were removed, dehydrated, stained with 15% modified Giemsa stain for 7 min, and then mounted on glass slides. Cells in 10 high power fields were counted and averaged for each sample. Migration index (MI) was calculated as the number of cells migrating toward the concentration gradients of chemokines divided by the number of cells migrating toward medium only as previously described (22). For pretreatment with PTX, NK cells (1 × 10⁶/mL) were either left intact or were treated for 2 h at 37°C with 100 ng/mL-activated PTX, as previously described (22). Only more than 95% viable cells were examined.

IL-2-activated NK cells were either left intact or incubated with various concentrations of GA, MMF, or DMF for 24 h. The cells were washed and incubated in a 96-well plate (v-bottom, 2 × 10⁵ cells per well), washed again, and resuspended in PBS buffer containing 0.1% sodium azide and 2% fetal bovine serum. Cells were first checked for viability with Trypan blue exclusion test, and only more than 95% viable cells were used in the assay. These viable cells were labeled with antibodies for 45 min in the dark at 4°C, washed twice, and examined in the flow cytometer (FACSC II, Becton-Dickinson Biosciences, San Jose, CA, USA). Markers were set according to the isotype control FITC- or PE-conjugated mouse IgG. More than 95% viable NK cells were also incubated at 4°C in the dark with FITC-conjugated anti-CD107a or FITC-conjugated IgG1 isotype control (Becton-Dickinson Pharmingen, San Diego, CA, USA). They were washed twice and examined in the flow cytometry. Gating was performed according to the isotype control. Analysis was done by FlowJo (Flow cytometry analysis software, Ashland, OR, USA).

More than 95% viable NK cells treated with 100 ng, 1 μg, or 10 μg GA for 24 h were lysed with ice-cold Non-idet P-40 lysis buffer containing 1% Non-idet P-40, 30 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethyl-sulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Sigma-Aldrich). The solutions were centrifuged at 13,000 × g for 15 min at 4°C to separate the cell lysates from the supernatants. Total protein concentration in the samples was determined using Bio-Rad Protein Assay (Bio-Rad, Uppsala, Sweden), and samples with 20-μg protein were used for each well in the gel. The samples were run on SDS-PAGE criterion gels and then electro transferred to PVDF-membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in TBS for 1 h, and then incubated overnight at 4°C with 1:250 dilution of polyclonal rabbit antihuman CCR10 or 1:8000 dilution of polyclonal rabbit anti-GADPH (Both from Abcam, Cambridge, UK). The membranes were washed four times in TBS-T-buffer, before incubation with HRP-conjugated secondary antibody (1:2500) (Bio-Rad) for 1 h at room temperature. Binding of antibodies to the target proteins was detected by secondary HRP-labeled antibodies and Super Signal West Pico Stable Peroxide Solution (Pierce, Rockford, IL, USA), using chemiluminescence film and CURIX 60 (Agfa HealthCare, Mortsel, Belgium).

Natural killer cells (1 × 10⁶/mL) were incubated with 100 ng, 1 μg, or 10 μg of GA, or with culture medium as a control for 24 h at 37°C in 5% CO₂ incubator. After incubation, the cells were harvested, and the cell suspensions were centrifuged at 1000 g for 8 min. Supernatants were collected and stored at −80°C until further analysis. Levels of chemokine were measured using ELISA kits from RayBiotech Inc. (Norcross, GA, USA). Color intensity was measured at 450 nm in a BioTek PowerWave XS plate reader. The standard curves and concentrations were calculated using Gen5 Data Analysis Software (BioTek Instruments, Winooski, VT, USA).

IL-2-activated NK cells (1 × 10⁶/mL) were left intact for 24 h. Supernatants were collected from these cells and were kept at −80°C until use. To detect the expression of Granzyme B (GrB), the cells (1 × 10⁶/mL) were incubated with either media alone or with 10 μg/mL of GA overnight. In addition, the cells treated as above (with or without GA) were preincubated with supernatants collected from activated NK cells either alone or with 1 μg/mL mouse antihuman CCL27 or mouse antihuman CCL28 (R&D systems), and as a control mouse IgG1.

To stain the intracellular GrB, cells were incubated with 10 μg/mL Brefeldin A (Sigma-Aldrich) for 4 h. They were then fixed with 4% paraformaldehyde for 15 min at 4°C and then washed twice with SAP buffer (PBS with 0.1% Saponin and 0.05% NaN3) before staining intracellularly with either PE-conjugated mouse antihuman Granzyme B or isotype control PE-conjugated mouse IgG antibody (both from ImmunoTools, Friesoythe, Germany) in the dark at 4°C for 45 min. Cells were washed with flow cytometric medium and resuspended with PBS in 5-mL tubes to perform flow cytometric analysis. Gating was done according to the PE-conjugated isotype control antibody. Analysis was done by FlowJo (flow cytometry analysis software, Ashland, OR, USA).

Significant values were generated using several tests. We used one-way ANOVA, two-way ANOVA with Bonferroni post hoc correction, or the Student’s t-test. A P value <0.05 was considered to be statistically significant.

Natural killer cells migrate into inflammatory sites aided by sets of chemokine receptors, which direct them toward the chemokines present at tumor growth sites. To this end, we investigated the expression of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, and CX₃CR1 on the surface of IL-2-activated NK cells after 24 h incubation with 1 or 10 μg/mL of GA. Only more than 90% viable cells as determined by Trypan blue exclusion test were used in this and subsequent assays. The reasons for choosing activated NK cells are due to the fact that at, inflammatory sites such as the tumor microenvironment, inflammatory molecules including cytokines and chemokines are released, which activate NK cells. In addition, activated NK cells migrate toward sites of inflammation much more efficiently than non-activated cells [reviewed in Ref. (12)].

From all the chemokine receptors examined, GA upregulated the expression of CCR1 and CCR10 on the surface of activated NK cells (Figure 1A). Also shown are histograms where CCR10 is expressed on activated NK cells after incubating overnight with 10 μg/mL of GA (Figure 1B). We focused the rest of this work on the expression of CCR10, since another drug used for treating MS patients, namely DMF also upregulates similar expression (also see Figure 4). To confirm the upregulation of CCR10 after GA stimulation, we performed immunoblot analysis. Results in Figure 1C demonstrate that overnight incubation with 0.1, 1, or 10 μg/mL of GA induced the expression of this receptor on activated NK cells, despite the fact that flow cytometric analysis did not show increase in expression of this receptor after incubation with 1 μg/mL of GA (Figure 1A).

Consequently, we examined the chemotaxis of activated NK cells treated for 24 h with 10 μg/mL GA toward various concentrations (ranging from 1 to 1000 ng/mL) of CCL27 and CCL28, the ligands for CCR10. Cells untreated with GA migrated with low intensity toward high concentrations of CCL27 (100 and 1000 ng/mL) and CCL28 (10 and 100 ng/mL), which could be due to the low number of CCR10 positive NK cells. However, cells treated with GA for 24 h migrated toward much lower concentrations of CCL27 and CCL28 (Figure 1D). When compared to untreated cells, the migration of cells toward the lower concentrations of these chemokines was enhanced by twofold to fivefold after treatment with GA, an enhancement that was statistically significant (Figure 1D). As a control, we also observed that these cells migrated toward the concentration gradients of CCL3, one of the ligands for CCR1 after treatment with GA (Figure 1D), which is corroborated with increased CCR1 expression on the surface of these cells after similar treatment.

We also investigated whether G proteins might be involved in mediating the chemotactic response. Consequently, activated NK cells were either left intact or were pretreated with PTX for 2 h, which intoxicates and inhibits the function of G_i/G_o (22). As shown in Figure 2A, NK cells migrated toward 10 ng/mL CCL27 after preincubating for 24 h with 10 μg/mL GA (P < 0.001, as compared to the control). This effect was inhibited after preincubating NK cells with PTX (P < 0.04). Similarly, 1 ng/mL of CCL28 induced the chemotaxis of NK cells pretreated overnight with GA (P < 0.04, as compared to the control), and this activity was abrogated upon pretreating the cells with PTX (P < 0.05, Figure 2B).

To evaluate whether GA might induce activities other than chemotaxis in NK cells, we performed the cytotoxicity assay. Results shown in Figure 3 demonstrate that incubating NK cells overnight with 10 μg/mL of this drug increased activated NK cell lysis of K562 cells (Figure 3A), or RAJI cells (Figure 3B). Because GA upregulates the expression of CCR10 on the surface of NK cells, we sought to demonstrate whether such expression might influence the cytolytic activity of these cells. NK cells either untreated or incubated with 10 μg/mL anti-CCR10, or as a control with isotype control IgG antibody, were examined for their ability to lyse tumor target cells in the NK cytotoxicity assay. We observed that anti-CCR10, and not the isotype control IgG, tended to inhibit NK cell lysis induced by GA of K562 (Figure 3A), but significantly inhibited lysis of RAJI cells (P < 0.05, comparing killing in the presence of anti-CCR10 to isotype control IgG, Figure 3B). Of note, cells treated overnight with GA and then incubated with isotype control IgG antibody also lysed K562 cells (Figure 2A) or RAJI cells (Figure 2B), but this did not reach statistical significance when compared to untreated cells incubated with the isotype control antibody.

Because anti-CCR10 inhibited GA-induced NK cell cytotoxicity, we entertained various possibilities to explain such activity. First, we sought to determine whether GA might induce the release of CCL27 or CCL28 from activated NK cells, and that these chemokines might facilitate NK cell cytolytic activity by binding CCR10. Our results demonstrate that IL-2-activated NK cells released an average of 280 pg/mL of CCL27 (Figure S2A in Supplementary Material), or 500 pg/mL CCL28 (Figure S2B in Supplementary Material). However, addition of various concentrations ranging from 0.1 to 10 μg/mL of GA to IL-2-activated NK cells did not significantly affect the levels of these chemokines, although a trend in increased release of CCL28 was noted after pretreatment with 10 μg/mL GA (Figure S2 in Supplementary Material).

Next, we examined whether GA or a combination of GA with CCL27 or CCL28 might induce the expression of CD107a on the surface of activated NK cells. Histograms of flow cytometric analysis showed a trend of increased CD107a expression after incubating the cells with GA, CCL27, or CCL28 (Figure S2C in Supplementary Material, left). Combining GA with CCL27 or CCL28 did not augment such expression when compared to cells incubated with GA alone (Figure S2C in Supplementary Material, right). When three or more experiments were performed, the trend of increased expression of CD107a was also noticed after incubating the cells with GA, CCL27, or CCL28. However, such enhancement did not reach significant levels after treating the cells with GA, any of the two chemokines, or GA plus the chemokines (Figure S2D in Supplementary Material).

To further investigate the plausible mechanisms of CCR10/chemokines axis, we measured the expression of Granzyme B in activated NK cells. For this, NK cells were either incubated with media alone or with 10 μg/mL of GA overnight, and the expression of Granzyme (GrB) was detected by flow cytometric analysis. Such treatment increased the percentages of cells expressing GrB from 6 to about 30% after pretreatment with the drug (Figure S3 in Supplementary Material). Supernatants collected from activated NK cells, when added to NK cells, also increased such expression to about 12%. The enhancement of GrB expression by the supernatants was lowered by pretreatment with anti-CCL28 to about 6%, but not with anti-CCL27, whereas the isotype control for the anti-CCL27 and anti-CCL28 also did not affect the activity of the supernatants (Figure S3 in Supplementary Material, upper panels). Addition of the supernatants to GA did not result in any synergy among their activities. However, anti-CCL28, and to a lesser extent anti-CCL27, reduced the effect of GA on GrB expression (Figure S3 in Supplementary Material, lower panels).

To determine whether other drugs or metabolites used to treat or have potential for treating MS or cancer patients might affect the expression of CCR10 on NK cells similar to GA, we investigated the effects of various drugs. We observed that none of the concentrations of vitamin D₃, its metabolite Calcipotriol, or FTY720 upregulated the expression of CCR10 on the surface of activated NK cells (not shown). In contrast, two different concentrations of MMF (1 and 100 μM) or the 100 μM concentration of DMF significantly upregulated the expression of CCR10 on the surfaces of NK cells (Figure 4A). MMF and the drug DMF also upregulated the expression of CXCR3, but not any other chemokine receptor examined (Figure 4A). Histograms of one representative experiment showed that 1 and 100 μM of MMF or 100 μM of DMF upregulated the expression of CCR10 on the surface of human IL-2-activated NK cells when compared to cells unstimulated with the drugs (Figure 4B). To corroborate the expression of CCR10 with functional activity, we performed the chemotaxis assay. Treatment of NK cells with MMF for 24 h enhanced their chemotaxis toward CCL27, CCL28, and CXCL10, the ligand for CXCR3 (Figure 4C). These results correlated well with increased expression of CCR10 and CXCR3 on NK cells after incubation with 1 or 100 μM MMF. Surprisingly, pretreatment of IL-2-activated NK cells with DMF did not increase their chemotaxis toward CCL27. However, their chemotaxis was increased toward 1 μg/mL of CCL28 or CXCL10 after overnight incubation with 100 μM DMF (Figure 4D).

We recently reported that MMF enhanced freshly isolated NK cell lysis of tumor cells (20). Because of the differences in the expression of NK cytotoxicity receptors among naive vs. activated NK cells, we sought to investigate whether MMF or DMF might enhance activated NK cell lysis of tumor cells. Pretreatment with either MMF or DMF enhanced IL-2-activated NK cell killing of K562 tumor cells (Figure 5A). However, only pretreatment with MMF, and not DMF, augmented activated NK cell lysis of the B cell lymphoma RAJI cells (Figure 5B). These results mirrored our recent findings showing that MMF, and not DMF, increased lysis of naive NK cells against RAJI cells (20).

Next, we asked whether CCR10 might play a role in MMF or DMF augmentation of NK cell cytotoxicity. Similar to its effect on GA-enhanced NK cell cytotoxicity, incubating activated NK cells with anti-CCR10 for 45 min abrogated the cytotoxicity induced by MMF or DMF against K562 cells (Figure 5A) or MMF-enhanced activated NK cell cytotoxicity against RAJI tumor cells (Figure 5B). This effect is not related to a general toxic effect of anti-CCR10 antibody as more than 95% of the cells were viable after treatment for 45 min with the antibodies.