Dendritic cells (DC) trigger activation and IFN-γ release by NK cells in lymphoid tissues, a process important for the polarization of Th1 responses. Little is known about the molecular signals that regulate DC-induced NK cell IFN-γ synthesis. In this study, we analyzed whether the interaction between Qa-1b expressed on DC and its CD94/NKG2A receptor on NK cells affects this process. Activation of DC using CpG-oligodeoxynucleotides in Qa-1b-deficient mice, or transfer of CpG-oligodeoxynucleotide-activated Qa-1b-deficient DC into wild-type mice, resulted in dramatically increased IFN-γ production by NK cells, as compared with that induced by Qa-1b-expressing DC. Masking the CD94/NKG2A inhibitory receptor on NK cells in wild-type mice similarly enhanced the IFN-γ response of these cells to Qa-1b-expressing DC. Furthermore, NK cells from CD94/NKG2A-deficient mice displayed higher IFN-γ production upon DC stimulation. These results demonstrate that Qa-1b is critically involved in regulating IFN-γ synthesis by NK cells in vivo through its interaction with CD94/NKG2A inhibitory receptors. This receptor-ligand interaction may be essential to prevent unabated cytokine production by NK cells during an inflammatory response.

Once activated, dendritic cells (DC)3 that have taken up and processed Ags in the periphery migrate to secondary lymphoid organs where they initiate immune responses. Although DC can present Ags to naive T cells, leading to the expansion of Ag-specific T cells, recent studies in mice have demonstrated that DC can also stimulate NK cells, leading to enhanced tumor cell killing (1) and IFN-γ release in vivo (2). NK cells are activated by DC through direct cell contact involving the formation of immunostimulatory synapses with polarized secretion of IL-12 toward NK cells (3). This process is further enhanced by other DC-derived cytokines, such as IL-2, IL-15, IL-18, and IFN-α (4, 5, 6, 7). NK cells, through their secretion of IFN-γ, TNF-α, and GM-CSF, in turn promote DC maturation, enhancing the ability of DC to prime CD8+ CTLs and CD4+ Th1 cells (8, 9). Thus, DC-NK interactions occurring early during an immune response can regulate downstream adaptive responses and function as a bridge between innate and adaptive immunity.

NK cells may interact with DC both in the periphery and in lymphoid tissues (10, 11). Studies in humans show that CD56brightKIR NK cells, which are abundant in lymph nodes (LN) and sites of chronic inflammation, express high levels of CD94/NKG2A and interact with DC (12, 13, 14). The CD94 glycoprotein forms heterodimers with C-type lectins of the NKG2 family (15). NKG2A contains an ITIM and delivers inhibitory signals (15, 16), whereas NKG2C and NKG2E associate with the DAP12 adaptor protein, which includes tyrosine-based activating motifs (ITAM) (17, 18). The inhibitory NKG2A molecule is the predominant isoform expressed by both human and murine NK cells and it has been suggested that the major function of CD94/NKG2A is to protect normal cells from NK cell-mediated killing (18, 19), whereas its role in regulating cytokine release is less clear. Qa-1b (or its human homolog HLA-E), the ligand for the CD94/NKG2A receptor, is a nonclassical MHC class I molecule with limited polymorphism that is widely expressed, albeit at lower levels than classical MHC I molecules (16, 20). Both Qa-1b and HLA-E predominantly bind nonameric peptides derived from the signal sequence of classical MHC class I molecules (21).

Although release of cytokines by NK cells and DC plays an important role in their reciprocal activation, the mechanisms that prevent unabated cytokine production by these cells remain unknown. In this study, we investigate the role of Qa-1b expressed on DC in modulating NK cell cytokine release. Our results show that the interaction between Qa-1b and its CD94/NKG2A receptor regulates the production of IFN-γ by NK cells both in vitro and in vivo.

C57BL/6 mice and DBA/2J mice were purchased from The Jackson Laboratory. DBA/2NCr mice were purchased from Charles Rivers Breeding Laboratories. C57BL/6 Qa-1b-deficient mice were described previously (22). All experiments involving animals were done in compliance with federal laws and protocols approved by Stanford University.

DC were generated by culturing bone marrow cells in IMDM (Invitrogen Life Technologies) supplemented with 10% FBS, 2 mM glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin, and 50 μM 2-ME in the presence of recombinant murine GM-CSF and IL-4 (both used at 10 ng/ml; PeproTech). After 5 days, cells were harvested and enriched using CD11c magnetic beads (Miltenyi Biotec), resulting in 99% pure DC. NK cells were isolated from naive spleens by depleting non-NK cells using a mixture of biotin-conjugated Abs against CD19, CD4 (L3T4), CD8a (Ly-2), CD5 (Ly-1), Gr-1 (Ly-6G/C), and Ter-119 and anti-biotin microbeads (Miltenyi Biotec), followed by a positive selection step using CD49b (DX5) magnetic beads (Miltenyi Biotec). The purity of enriched splenic NK cells was >95%. For the experiment shown in Fig. 1 C, NK cells were sorted into CD3NK1.1+CD94dim and CD3NK1.1+CD94bright cells using a FACSVantage (BD Pharmingen). Sorted NK cells (95–99% pure) were cultured for 4 days in medium containing recombinant murine IL-15 (20 ng/ml; PeproTech) before being washed extensively and used for in vitro assays. Cocultures were established for 18 h using enriched bulk NK cells or sorted NK cell subsets, with DC at 5 × 104–1 × 105 cells/well in 96-well plates at a 1:1 cell ratio. Unmethylated CpG oligonucleotide motifs (CpG-ODN 1826) (5′-TCCATGACGTTCCTGACGTT-3′) were used at a concentration of 5 μg/ml and were added to cells at the time of coculture. Where indicated, cells were cultured separated by a Transwell insert (Nalge Nunc International).

FIGURE 1.

Reciprocal activation of DC and NK cells in response to CpG-ODN stimulation. A, Qa-1b expression on BM-DC cultured alone (dashed line), in the presence of CpG-ODN (black line), or with autologous splenic NK cells plus CpG-ODN (gray line). Filled histogram shows isotype control staining. B, Anti-CD94 (left histogram plot), anti-NKG2A/C/E (middle plot), and anti-NKG2A (right plot) staining patterns on splenic NK cells cultured with CpG-ODN alone (gray line) or with DC in the presence of CpG-ODN (black line). Dashed line in the left histogram plot shows anti-CD94 staining of NK cells cultured with DC plus CpG-ODN but separated by a Transwell insert. Filled histograms show staining with the corresponding isotype control. C, Anti- CD94 (left histogram), anti-NKG2A/C/E (middle histogram), and anti-NKG2A (right histogram) staining patterns on NK cells sorted into NK1.1+ CD3CD94dim or CD94bright subsets before culture with CpG-ODN alone (gray line) or with DC plus CpG-ODN (black line). Dashed line in the left histogram plots shows CD94 staining of NK cells cultured with DC plus CpG-ODN but separated by a Transwell insert. Filled histogram shows staining with isotype control. D, CD69 expression on splenic NK cells cultured with DC (gray line) or DC plus CpG-ODN (black line). Filled histogram shows isotype control staining. E, Cytokine production in DC-NK cocultures in the presence or absence of CpG-ODN. IFN-γ (left) and IL-12p70 (right) levels in cell-free culture supernatants were assessed by ELISA. TW refers to cultures in which NK cells and DC were separated by a Transwell insert. Error bars, Mean ± SD. Asterisks, p < 0.05.

FIGURE 1.

Reciprocal activation of DC and NK cells in response to CpG-ODN stimulation. A, Qa-1b expression on BM-DC cultured alone (dashed line), in the presence of CpG-ODN (black line), or with autologous splenic NK cells plus CpG-ODN (gray line). Filled histogram shows isotype control staining. B, Anti-CD94 (left histogram plot), anti-NKG2A/C/E (middle plot), and anti-NKG2A (right plot) staining patterns on splenic NK cells cultured with CpG-ODN alone (gray line) or with DC in the presence of CpG-ODN (black line). Dashed line in the left histogram plot shows anti-CD94 staining of NK cells cultured with DC plus CpG-ODN but separated by a Transwell insert. Filled histograms show staining with the corresponding isotype control. C, Anti- CD94 (left histogram), anti-NKG2A/C/E (middle histogram), and anti-NKG2A (right histogram) staining patterns on NK cells sorted into NK1.1+ CD3CD94dim or CD94bright subsets before culture with CpG-ODN alone (gray line) or with DC plus CpG-ODN (black line). Dashed line in the left histogram plots shows CD94 staining of NK cells cultured with DC plus CpG-ODN but separated by a Transwell insert. Filled histogram shows staining with isotype control. D, CD69 expression on splenic NK cells cultured with DC (gray line) or DC plus CpG-ODN (black line). Filled histogram shows isotype control staining. E, Cytokine production in DC-NK cocultures in the presence or absence of CpG-ODN. IFN-γ (left) and IL-12p70 (right) levels in cell-free culture supernatants were assessed by ELISA. TW refers to cultures in which NK cells and DC were separated by a Transwell insert. Error bars, Mean ± SD. Asterisks, p < 0.05.

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For staining of cells for flow cytometry, the following fluorochrome-conjugated mAbs were used: anti-NK1.1 (PK136), anti-CD3 (145-2C11), anti-CD11c (HL3), anti-CD69 (H1.2F3), anti-CD94 (18d3), anti-NKG2A/C/E (20d5), anti-NKG2A (16a11), anti-Qa-1b (6A8.6F10.1A6), anti-CD49b (DX5) and anti-IFN-γ (XMG1.2) and the corresponding isotype control Abs (all from BD Pharmingen and used at 1 μg/106 cells). Flow cytometry was performed using a FACSCalibur (BD Pharmingen) and CellQuest software was used for analysis.

IL12p70 and IFN-γ concentrations in cell-free culture supernatants were determined using murine IL12p70 or IFN-γ ELISA duo sets (R&D Systems) with a sensitivity of detection of 7 and 30 pg/ml, respectively.

To assess the effect of CpG-ODN-induced immune activation on NK cells in vivo, mice were injected s.c. in the right footpad with 5 μg of CpG-ODN 1826 and 48 h later popliteal LN were harvested. To examine NK cell activation induced by DC in vivo, CpG-ODN-treated BM-DC were washed twice with PBS and 2 × 106 wild-type (WT) DC were injected s.c. in the right footpad in a 50-μl volume, whereas 2 × 106 Qa-1b-deficient DC were injected s.c. in the left footpad. Forty-eight hours later, popliteal LN were harvested and single-cell suspensions were treated with PMA (10 ng/ml; Sigma-Aldrich) and ionomycin (0.5 μM; Sigma-Aldrich) for 4 h, with brefeldin A (10 μg/ml; Sigma-Aldrich) added during the last 2 h before surface staining for CD3 and NK1.1 or DX5, followed by intracellular IFN-γ staining using a Cytofix/Cytoperm kit (BD Pharmingen). PMA and ionomycin pretreatment did not induce IFN-γ expression in NK cells from control LN, and when in vivo-activated NK cells were analyzed in parallel with or without addition of PMA/ionomycin, we observed that brefeldin A treatment without PMA/ionomycin before intracellular staining resulted in a slightly lower frequency of IFN-γ-positive NK cells overall (data not shown). Isotype-matched control mAbs were included in all experiments. For in vivo CD94/NKG2A-blocking experiments, 50 μg of purified anti-mouse NKG2A/C/E Ab (clone 20d5; Serotec) or the corresponding isotype control (rat IgG2a; Serotec) was injected into the right footpad 5 h before injection of 2 × 106 DC in the same region. DC migration to LN was analyzed by injecting 2 × 106 CFSE-labeled (Molecular Probes) DC s.c. into the right footpad and, after 48 h, popliteal LN cells were stained with anti-CD11c mAb and then analyzed by flow cytometry.

Where appropriate, data are expressed as the mean ± SD. Student’s t test was used to compare the differences between two means. For each test, p < 0.05 was considered statistically significant, indicated by an asterisk (∗).

We first assessed the phenotypic changes that take place in cocultures of bone marrow-derived DC (BM-DC) and freshly isolated splenic NK cells from C57BL/6 mice, in the presence or absence of CpG-ODN, which activate DC and drive Th1 polarization through binding to TLR9 (23). Interestingly, Qa-1b expression was up-regulated on DC upon CpG-ODN treatment in vitro and no further up-regulation was noted in the presence of NK cells (Fig. 1,A). Addition of CpG-ODN to DC-NK cell cocultures induced up-regulation of the inhibitory receptor specific for Qa-1b (i.e., CD94/NKG2A heterodimer) on NK cells, which was dependent on NK cell contact with DC (Fig. 1, B and C).

CD94 pairs with members of the NKG2 family and two distinct NK cell populations can be identified based on the CD94 expression level (i.e., a CD94dim and CD94bright NK cell subsets). These subsets are present at equal frequencies in blood and other tissues (e.g., spleen, LN, bone marrow, and liver; data not shown). Most CD94bright NK cells are NKG2A positive, whereas the CD94dim subset is NKG2A negative and also lacks detectable NKG2A/C/E (Fig. 1,C), suggesting that the inhibitory CD94/NKG2A heterodimer is the predominant form present on mouse NK cells in accordance with a previous report (18). By sorting NK cells into CD94dim and CD94bright subsets before DC coculture, we noted that the CD94/NKG2A heterodimer is primarily induced on the CD94bright subset, while CD94 up-regulation on the CD94dim subset is not associated with increased NKG2A levels (Fig. 1 C).

As shown in Fig. 1, D and E, CpG-ODN-treated DC, but not immature DC, induced CD69 up-regulation and triggered IFN-γ release by NK cells in vitro. CpG-ODN-treated DC cultured alone did not produce IFN-γ as assessed by ELISA (Fig. 1,E), and intracellular staining demonstrated that the IFN-γ produced in the DC-NK cell cocultures was derived almost exclusively from NK cells (data not shown). Conversely, IL-12 synthesis by DC upon CpG-ODN treatment was significantly enhanced if the cells were stimulated in the presence of NK cells (Fig. 1,E). Although low levels of NK cell-derived IFN-γ were produced when NK cells and DC were cultured separately using a Transwell insert, significantly higher levels of this cytokine were produced when the cells were in direct contact (Fig. 1 E), suggesting that optimal activation of NK cells requires direct contact with DC.

The observations that CD94/NKG2A expression is up-regulated on NK cells upon contact with activated DC and that Qa-1b is increased on CpG-ODN-treated DC suggest that an interaction between this receptor-ligand pair may play a role in regulating DC-NK cell cross-activation. To determine whether Qa-1b is involved in the regulation of IFN-γ synthesis by NK cells in vivo, we performed experiments in Qa-1b-deficient mice, which have a similar frequency of NK cells and a similar CD94/NKG2A expression pattern as WT C57BL/6 mice in LN and spleen (Ref. 22 and data not shown). Furthermore, NK cells from Qa-1b-deficient mice produced similar levels of IFN-γ as WT NK cells upon cross-linking NK1.1 in vitro (data not shown). Nevertheless, compared with NK cells from WT mice, NK cells from Qa-1b-deficient mice responded more vigorously to CpG-ODN stimulation in vivo as indicated by both a higher frequency of IFN-γ+ NK cells (Figs. 2, A and B) and greater IFN-γ synthesis per cell, reflected by a higher IFN-γ mean fluorescence intensity (Fig. 2 C). Taken together, these data suggest that Qa-1b limits IFN-γ synthesis by NK cells.

FIGURE 2.

Enhanced NK cell-derived IFN-γ production in Qa-1b-deficient mice. A, C57BL/6 WT or Qa-1b -deficient mice were injected with 5 μg of CpG-ODN or PBS s.c. in the right footpad. After 48 h, popliteal LN draining the injection site or contralateral LN were harvested and cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage and mean fluorescence intensity (in parentheses) of IFN-γ+ cells of total NK1.1+CD3 cells. One representative experiment is shown of a total of five mice analyzed in two separate experiments. B, Percentage of IFN-γ+ NK cells in LN of WT or Qa-1b-deficient (Qa-1−/−) mice 48 h after injection of CpG-ODN (CpG) or PBS. CL refers to contralateral LN opposite to the CpG-ODN injection site. C, Mean fluorescence intensity of IFN-γ staining of LN NK cells derived from WT or Qa-1b-deficient (Qa-1−/−) mice 48 h after injection of CpG-ODN (CpG) or PBS. CL refers to contralateral LN opposite to the CpG-ODN injection site. Data represent the mean ± SD of data from five mice analyzed in two separate experiments. Asterisks, p < 0.05.

FIGURE 2.

Enhanced NK cell-derived IFN-γ production in Qa-1b-deficient mice. A, C57BL/6 WT or Qa-1b -deficient mice were injected with 5 μg of CpG-ODN or PBS s.c. in the right footpad. After 48 h, popliteal LN draining the injection site or contralateral LN were harvested and cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage and mean fluorescence intensity (in parentheses) of IFN-γ+ cells of total NK1.1+CD3 cells. One representative experiment is shown of a total of five mice analyzed in two separate experiments. B, Percentage of IFN-γ+ NK cells in LN of WT or Qa-1b-deficient (Qa-1−/−) mice 48 h after injection of CpG-ODN (CpG) or PBS. CL refers to contralateral LN opposite to the CpG-ODN injection site. C, Mean fluorescence intensity of IFN-γ staining of LN NK cells derived from WT or Qa-1b-deficient (Qa-1−/−) mice 48 h after injection of CpG-ODN (CpG) or PBS. CL refers to contralateral LN opposite to the CpG-ODN injection site. Data represent the mean ± SD of data from five mice analyzed in two separate experiments. Asterisks, p < 0.05.

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To assess the importance of Qa-1b expressed by activated DC in the regulation of IFN-γ released by NK cells, we generated BM-DC from either wild type or Qa-1b-deficient mice. Both populations responded similarly to CpG-ODN stimulation in vitro based on their production of IL-12 (data not shown). We then cultured freshly isolated NK cells with WT or Qa-1b-deficient DC in the presence of CpG-ODN and assessed IFN-γ production by NK cells using intracellular staining (Fig. 3, A and B) and ELISA (Fig. 3,C). In both instances, Qa-1b-deficient DC triggered greater IFN-γ synthesis by NK cells than WT DC. To determine whether this difference is also seen in vivo, we studied local responses in LN of mice immunized with CpG-ODN-activated BM-DC. In the first set of experiments, we immunized mice with CFSE-labeled DC s.c. in the footpad. The injected DC migrated to local LN, as CFSE-labeled DC were detected in LN draining the injection site but not in nondraining LN (data not shown). In addition, we observed an enlargement and increased cellularity in DC-draining LN, with higher numbers of NK cells, T cells, and DC compared with either control LN (i.e., PBS injected) or contralateral LN (Table I). Moreover, based on CD69 expression and intracellular IFN-γ staining, we noted that NK cells in DC-draining LN were activated, whereas NK cells derived from either control or contralateral LN remained negative for IFN-γ and CD69 (data not shown). These first set of observations indicated that s.c. injected mature DC induced local immune responses in LN, including NK cell recruitment and NK cell activation, in accordance with a previous report (2).

FIGURE 3.

Qa-1b-deficient DC induce greater IFN-γ production by NK cells than WT DC in vitro. A, Freshly isolated NK cells plus CpG-ODN were cultured alone (control) or in the presence of WT or Qa-1b-deficient DC. After 18 h, cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage of IFN-γ+ cells of total NK cells. One representative experiment is shown from duplicate wells analyzed in two separate experiments. B, Percentage of IFN-γ+ NK cells plus CpG-ODN cultured alone (NK) or in cocultures with WT DC or Qa-1b-deficient DC (Qa-1−/− DC). C, IFN-γ production by WT DC cultured alone, WT DC plus NK cells (WTDC NK). Qa-1b-deficient DC alone (Qa-1−/− DC), Qa-1b-deficient DC plus NK cells (Qa-1−/− DC NK), or NK cells alone. IFN-γ levels in cell-free culture supernatants were assessed by ELISA. Data represent the mean ±SD from duplicate wells analyzed in two separate experiments. Asterisks, p < 0.05.

FIGURE 3.

Qa-1b-deficient DC induce greater IFN-γ production by NK cells than WT DC in vitro. A, Freshly isolated NK cells plus CpG-ODN were cultured alone (control) or in the presence of WT or Qa-1b-deficient DC. After 18 h, cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage of IFN-γ+ cells of total NK cells. One representative experiment is shown from duplicate wells analyzed in two separate experiments. B, Percentage of IFN-γ+ NK cells plus CpG-ODN cultured alone (NK) or in cocultures with WT DC or Qa-1b-deficient DC (Qa-1−/− DC). C, IFN-γ production by WT DC cultured alone, WT DC plus NK cells (WTDC NK). Qa-1b-deficient DC alone (Qa-1−/− DC), Qa-1b-deficient DC plus NK cells (Qa-1−/− DC NK), or NK cells alone. IFN-γ levels in cell-free culture supernatants were assessed by ELISA. Data represent the mean ±SD from duplicate wells analyzed in two separate experiments. Asterisks, p < 0.05.

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Table I.

Recruitment of immune cells to LN after DC transfera

ControlDC-draining LNContralateral LN
Total cells (×1060.68 ± 0.3 5.9 ± 1.4 0.74 ± 0.3 
NK cells (×1032.72 ± 1.3 58.1 ± 22 2.4 ± 1.0 
T cells (×1053.8 ± 1.6 21.8 ± 6.1 3 ± 0.8 
DC (×1033.28 ± 0.7 30 ± 2.5 4.25 ± 0.8 
ControlDC-draining LNContralateral LN
Total cells (×1060.68 ± 0.3 5.9 ± 1.4 0.74 ± 0.3 
NK cells (×1032.72 ± 1.3 58.1 ± 22 2.4 ± 1.0 
T cells (×1053.8 ± 1.6 21.8 ± 6.1 3 ± 0.8 
DC (×1033.28 ± 0.7 30 ± 2.5 4.25 ± 0.8 
a

Data show the mean ± SD of total LN cells (n = 14), NK cells (n = 14), T cells (n = 14), and DC (n = 3) numbers observed in LN from mice injected with PBS (control) or 2 × 106 CpG-ODN-activated DC. Cell numbers were determined by analyzing NK1.1+CD3 cells (for NK cells), NK1.1CD3+ cells (for T cells), and CD11c+NK1.1 cells (for DC). DC-draining LN refers to LN draining the injection site. Contralateral LN refers to LN contralateral to the injection site.

Importantly, both WT and Qa-1b-deficient DC could be traced to LN draining the injection site but not to contralateral LN (Fig. 4,A). Knowing that DC-induced NK cell activation is confined to local LN, we then injected WT mice in the left footpad with Qa-1b-deficient DC and simultaneously in the right footpad with WT DC. We observed a significantly higher frequency of IFN-γ+ NK cells in the LN draining the Qa-1b-deficient DC injection site (Fig. 4, B and C), and these NK cells produced more IFN-γ per cell than WT NK cells (Fig. 4,D). The NKG2A+ NK cell subset consistently produced higher amounts of IFN-γ than the NKG2A subset after injection of DC (Fig. 4 E), and Qa-1b-deficient DC induced more IFN-γ in NKG2A+ than NKG2A NK cells. It should be noted that NKG2A NK cells also displayed an increase in IFN-γ levels upon stimulation with Qa-1b-deficient DC, although to a lesser extent than the NKG2A+ NK cells. Altogether, these observations suggest that Qa-1b expressed by DC modulates NK cell IFN-γ synthesis in LN.

FIGURE 4.

Increased IFN-γ synthesis induced by Qa-1b-deficient DC. A, 2 × 106 CFSE-labeled CpG-ODN-treated DC derived from WT or Qa-1b-deficient mice were injected s.c. in the right footpad, and 48 h later DC draining or contralateral LN were harvested and the number of CFSE+CD11c+ cells was analyzed by flow cytometry. Numbers in quadrants are an average of two mice analyzed. B, Mice were injected s.c. in the right footpad with 2 × 106 WT CpG-ODN-treated DC and in the left footpad with 2 × 106 Qa-1b-deficient CpG-ODN-treated DC. After 48 h, popliteal LN were harvested and LN cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage and mean fluorescence intensity (in parentheses) of IFN-γ+ cells of total NK1.1+CD3 cells. One representative experiment is shown from a total of five mice analyzed. C, Percentage of IFN-γ+ NK cells in LN of mice injected with CpG-treated DC derived from WT DC or Qa-1b-deficient (Qa-1−/− DC) mice. D, IFN-γ-mean fluorescence intensity in LN NK cells of mice injected with CpG-treated DC derived from WT DC or Qa-1b-deficient (Qa-1−/− DC) mice. Data represent the mean ± SD of five mice analyzed in two experiments. Asterisks, p < 0.05. E, Mice were injected s.c. in the right footpad with 2 × 106 WT CpG-ODN-treated DC and in the left footpad with 2 × 106 Qa-1b-deficient CpG-ODN-treated DC. After 48 h, popliteal LN were harvested and LN cells were stained for CD3, NK1.1, NKG2A, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage and mean fluorescence intensity (in parentheses) of IFN-γ+ cells of total NKG2ANK1.1+CD3 cells (upper left quadrants) or NKG2A+NK1.1+CD3 cells (upper right quadrants) in one representative experiment.

FIGURE 4.

Increased IFN-γ synthesis induced by Qa-1b-deficient DC. A, 2 × 106 CFSE-labeled CpG-ODN-treated DC derived from WT or Qa-1b-deficient mice were injected s.c. in the right footpad, and 48 h later DC draining or contralateral LN were harvested and the number of CFSE+CD11c+ cells was analyzed by flow cytometry. Numbers in quadrants are an average of two mice analyzed. B, Mice were injected s.c. in the right footpad with 2 × 106 WT CpG-ODN-treated DC and in the left footpad with 2 × 106 Qa-1b-deficient CpG-ODN-treated DC. After 48 h, popliteal LN were harvested and LN cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage and mean fluorescence intensity (in parentheses) of IFN-γ+ cells of total NK1.1+CD3 cells. One representative experiment is shown from a total of five mice analyzed. C, Percentage of IFN-γ+ NK cells in LN of mice injected with CpG-treated DC derived from WT DC or Qa-1b-deficient (Qa-1−/− DC) mice. D, IFN-γ-mean fluorescence intensity in LN NK cells of mice injected with CpG-treated DC derived from WT DC or Qa-1b-deficient (Qa-1−/− DC) mice. Data represent the mean ± SD of five mice analyzed in two experiments. Asterisks, p < 0.05. E, Mice were injected s.c. in the right footpad with 2 × 106 WT CpG-ODN-treated DC and in the left footpad with 2 × 106 Qa-1b-deficient CpG-ODN-treated DC. After 48 h, popliteal LN were harvested and LN cells were stained for CD3, NK1.1, NKG2A, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage and mean fluorescence intensity (in parentheses) of IFN-γ+ cells of total NKG2ANK1.1+CD3 cells (upper left quadrants) or NKG2A+NK1.1+CD3 cells (upper right quadrants) in one representative experiment.

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To determine whether the CD94/NKG2A receptor is involved in regulating NK cell IFN-γ release through its interaction with Qa-1b expressed on DC, we evaluated the effect of blocking this receptor in vivo. We reasoned that if CD94/NKG2A has an inhibitory function on NK cells, masking this receptor should enhance cytokine production. C57BL/6 mice were injected s.c. with an Ab that reportedly masks CD94/NKG2A and thereby reverses the inhibition of NK cell cytotoxicity against Qa-1b-expressing target cells in vitro (18). Five hours later, the mice were injected in the same area with CpG-ODN-matured DC. As shown in Fig. 5, masking the CD94/NKG2A receptor in vivo markedly enhanced DC-induced IFN-γ responses by NK cells, compared with mice treated with the isotype control Ab. In contrast, mice injected with PBS or Ab alone showed no or negligible IFN-γ synthesis by NK cells. These results indicate that the interaction between Qa-1b molecules on DC and CD94/NKG2A on NK cells regulates NK cell IFN-γ synthesis in vivo. Furthermore, the observation that NK cells from DBA/2J mice, which are naturally CD94/NKG2A deficient, displayed higher IFN-γ synthesis in response to DC stimulation compared with NK cells from CD94/NKG2A-expressing substrains (Fig. 6) further supports the role of CD94/NKG2A in the regulation of DC-induced IFN-γ synthesis by NK cells in vivo.

FIGURE 5.

Enhanced IFN-γ production upon NKG2A/C/E masking. A, C57BL/6 mice were injected in the right footpad with 50 μg of anti-NKG2A/C/E mAb or with the corresponding isotype control Ab. Five hours later, PBS or 2 × 106 CpG-ODN-treated DC were injected into the same area. After 48 h, popliteal LN draining the site of injection were harvested and cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote percentage of IFN-γ+ cells of total NK1.1+CD3 cells. One representative experiment is shown of a total of five mice analyzed. B, Percentage of IFN-γ+ NK cells in control (PBS injected) and DC draining LN of mice pretreated with anti-NKG2A/C/E Ab or an isotype control Ab. Data are the mean ± SD of results obtained from a total of five mice studied in two separate experiments. Asterisk, p < 0.05.

FIGURE 5.

Enhanced IFN-γ production upon NKG2A/C/E masking. A, C57BL/6 mice were injected in the right footpad with 50 μg of anti-NKG2A/C/E mAb or with the corresponding isotype control Ab. Five hours later, PBS or 2 × 106 CpG-ODN-treated DC were injected into the same area. After 48 h, popliteal LN draining the site of injection were harvested and cells were stained for CD3, NK1.1, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote percentage of IFN-γ+ cells of total NK1.1+CD3 cells. One representative experiment is shown of a total of five mice analyzed. B, Percentage of IFN-γ+ NK cells in control (PBS injected) and DC draining LN of mice pretreated with anti-NKG2A/C/E Ab or an isotype control Ab. Data are the mean ± SD of results obtained from a total of five mice studied in two separate experiments. Asterisk, p < 0.05.

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FIGURE 6.

Enhanced NK cell-derived IFN-γ production in CD94-deficient mice. A, DBA/2NCr mice (CD94/NKG2A/C/E+) or DBA/2J (CD94/NKG2A/C/E) mice were injected with 2 × 106 CpG-ODN-treated DC (derived from DBA/2NCr mice) or PBS s.c. in the right footpad. After 48 h, popliteal LN draining the injection site were harvested and cells were stained for CD3, DX5, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage of IFN-γ+ cells of total DX5+CD3 cells. One representative experiment is shown from a total of three mice analyzed. B, Percentage of IFN-γ+ NK cells in LN of DBA/2NCr mice (CD94/NKG2A/C/E+) or DBA/2J (CD94/NKG2A/C/E) mice 48 h after injection of CpG-DC (DC) or PBS. Data represent the mean ± SD of data from three mice analyzed. Asterisks, p < 0.05.

FIGURE 6.

Enhanced NK cell-derived IFN-γ production in CD94-deficient mice. A, DBA/2NCr mice (CD94/NKG2A/C/E+) or DBA/2J (CD94/NKG2A/C/E) mice were injected with 2 × 106 CpG-ODN-treated DC (derived from DBA/2NCr mice) or PBS s.c. in the right footpad. After 48 h, popliteal LN draining the injection site were harvested and cells were stained for CD3, DX5, and intracellular IFN-γ and analyzed by flow cytometry. Numbers in upper quadrants denote the percentage of IFN-γ+ cells of total DX5+CD3 cells. One representative experiment is shown from a total of three mice analyzed. B, Percentage of IFN-γ+ NK cells in LN of DBA/2NCr mice (CD94/NKG2A/C/E+) or DBA/2J (CD94/NKG2A/C/E) mice 48 h after injection of CpG-DC (DC) or PBS. Data represent the mean ± SD of data from three mice analyzed. Asterisks, p < 0.05.

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The reciprocal stimulation of DC and NK cells is well documented (1, 24, 25). It is also widely accepted that NK cell activity is regulated by a balance of signals mediated through a variety of activating and inhibitory NK cell surface receptors that control both cytokine and cytotoxic responses (26). However, it is technically difficult to assess the physiological effect of a given inhibitory MHC class I ligand on NK cell cytokine responses in vivo. This is perhaps particularly true for Qa-1b, which forms a ligand for inhibitory CD94/NKG2A receptors only when it presents peptides primarily derived from other MHC class I molecules (21). Such complexes are likely gradually increased on target cells during an ongoing immune response involving IFN-γ production, which is a well-known inducer of MHC class I synthesis, including Qa-1b (27, 28).

Because NK cells are prominent producers of IFN-γ during early inflammatory responses (29, 30), we hypothesized that Qa-1b molecules on activated DC are involved in the modulation of NK cell-derived IFN-γ synthesis through their interaction with CD94/NKG2A receptors on NK cells. To test this hypothesis, we used CpG-ODN to activate DC derived from C57BL/6 WT or Qa-1b-deficient mice and detected similar levels of IL12 p70 secretion in vitro. In addition, such activated DC induced IFN-γ synthesis by NK cells, but optimal stimulation of NK cells was dependent on direct contact with DC. In turn, NK cells promoted further DC maturation as reflected by enhanced IL-12 p70 synthesis by DC in vitro. This was particularly evident when using CD94bright NK cells (data not shown), suggesting that this subset may correspond to the well-described CD56bright NK cell subset in human LN (12).

Because NK cells enhance IL-12 synthesis in DC, and such DC induce further IFN-γ synthesis by NK cells, mechanisms that prevent excessive accumulation of inflammatory mediators are essential to prevent immune-mediated tissue destruction. In vitro, IFN-γ production by NK cells was higher in cocultures with Qa-1b-deficient DC, compared with WT DC. Moreover, NK cell IFN-γ responses were augmented in vivo in CpG-ODN-treated Qa-1b-deficient mice, suggesting that Qa-1b is a key regulator of the amplitude of NK cell cytokine production. NK cells in lymphoid organs from Qa-1b- deficient mice were present at similar frequencies as C57BL/6 WT mice and displayed no differences in phenotype or activation state, suggesting that the enhanced NK cell IFN-γ production observed upon CpG-ODN treatment was due to a lack of inhibitory signals delivered upon Qa-1b engagement, rather than a particular property of NK cells in Qa-1b-deficient mice. In addition, a previous study showed that the latter mice have no detectable defects in the development of CD4+ and CD8+ T cells, but display enhanced secondary CD4+ T cell responses after infection or immunization, due in part to the absence of Qa-1b-restricted suppressor CD8+ T cells (22). The higher levels of NK cell-derived IFN-γ in these mice observed in the present study may also contribute to their enhanced T cell responses.

NK-DC-T cell interactions have been demonstrated in mouse LN, and cytokines released by NK cells may influence adaptive immune responses triggered locally (11). We found that s.c. injected CpG-ODN-activated DC trafficked selectively to the draining LN where they both recruited and activated NK cells in accordance with a previous report (2). Both WT and Qa-1b-deficient DC migrated to local LN and induced NK cell recruitment to a similar degree. Notably, NK cell IFN-γ production was significantly enhanced in mice injected with Qa-1b-deficient DC compared with mice injected with WT DC. The NKG2A+ NK cell subset consistently produced higher levels of IFN-γ as compared with the NKG2A subset when stimulated by DC in vivo, and these levels were further enhanced in response to Qa-1b-deficient DC. Unexpectedly, NKG2A NK cells also displayed a slight increase in IFN-γ synthesis upon Qa-1b−/− DC injection. It is possible that IFN-γ derived from NKG2A+ cells enhanced the production of this cytokine by NKG2A cells, since IFN-γ is known to activate NK cells (31). IFN-γ production was also enhanced in C57BL/6 mice that were pretreated with an Ab blocking CD94/NKG2A and in DBA/2J mice, which are naturally deficient in CD94/NKG2A, confirming the ability of this receptor to tune NK cell IFN-γ synthesis. To our knowledge, these results provide the first experimental evidence that DC-NK cross-talk is modulated by CD94-NKG2A interactions in vivo.

Recent reports indicate that CD94/NKG2A is expressed by a subset of CD3+NK1.1+ cells, that it can be induced on CD8+T cells, and that the cytotoxic activity mediated by these cells is regulated via CD94/NKG2A (32, 33, 34). In the current study, we observed an increased frequency of IFN-γ+CD3+NK1.1+ cells in LN of DC-injected mice, whereas CD3+NK1.1 cells remained IFN-γ negative (data not shown). The frequency of IFN-γ+CD3+NK1.1+ cells was an order of magnitude lower as compared with IFN-γ+ NK cells. Nonetheless, we observed a greater number of IFN-γ+ cells among NK1.1+ T cells after Ab-mediated masking of CD94/NKG2A in vivo, suggesting that, similar to NK cells, IFN-γ synthesis by NK1.1+ T cells is regulated through CD94/NKG2A signaling in vivo.

Our results show that even in the absence of detectable IFN-γ BM-DC express increased levels of Qa-1b upon CpG-ODN treatment, which was not further up-regulated in the presence of NK cells. It is possible that initial CpG-ODN-induced Qa-1b up-regulation on DC does not involve presentation of inhibitory signal peptides, thus allowing NK cells to produce IFN-γ without significant down-regulation through CD94/NKG2A receptors. However, NK cell-derived IFN-γ would be expected to expand the available pool of MHC class I signal peptides and thus the levels of protective Qa-1b complexes on the DC surface. As a consequence, we hypothesize that the amount of IFN-γ released by NK cells upon DC encounter varies with time and is initially dependent on DC-derived cytokines (e.g., IL-12, IL15, IL-18) and various costimulatory molecules expressed by DC that interact with activating NK cell surface receptors (3, 6, 7, 25, 35). Thereafter, IFN-γ-mediated up-regulation of protective Qa-1b molecules on DC would be expected to dampen NK cell IFN-γ release. Such cross-talk between NK cells and DC could modulate the amplitude of an inflammatory response against an invading pathogen and may help explain why certain viruses causing persistent infections (e.g., human CMV and certain mouse viruses) carry their own HLA-E/Qa-1b-binding peptides which potentially have evolved to dampen the magnitude of early IFN-γ release by NK cells (27). Any delay in the activation of this “switch-off” mechanism (e.g., by anti-NKG2A Ab treatment) would be expected to enhance NK cell IFN-γ release, leading to greater DC activation and more potent protective immunity. These events may also impact the type and magnitude of downstream adaptive immune responses to pathogens, as previously suggested (8, 36).

We thank Lorna Torentino for assistance with cell sorting, Donna Jones for secretarial assistance, and Claudia Benike for critically reviewing this manuscript.

The authors have no financial conflict of interest.

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 was supported by National Institutes of Health Grants HL57443, AR051748, and AI055468.

3

Abbreviations used in this paper used in this paper: DC, dendritic cell; ODN, oligodeoxynucleotide; LN, lymph node; BM-DC, bone marrow-derived DC; WT, wild type.

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