NK cells have been proposed to be an initial source of IFN-γ that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27high NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27high NK cell subset in the draining LN was dependent on host IFN-γ and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-γ-dependent mechanism. Thus, our results identify that CD27high NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-γ in coordinating with DC migration.
Natural killer cells recognize self-MHC molecules and ligands on stressed, transformed, or infected cells, thereby integrating signals transduced by various inhibitory and activating receptors, respectively (1, 2, 3). Activated NK cells kill target cells or produce inflammatory cytokines such as IFN-γ and therefore represent a primary arm of the innate immune response (4, 5). In addition to these effector activities, a series of studies in both humans and mice reveal that NK cells also influence adaptive immune responses by modulating dendritic cell (DC)2 function (6, 7, 8). In vitro coculture of NK cells with DC resulted in NK cell activation that was dependent on both soluble factors and cell-cell contact (9, 10, 11). Reciprocally, it has also been shown that activated NK cells kill immature DC or promote DC maturation (9, 10, 11, 12). Indeed, a previous study reported that NK cells contribute to the maintenance of CD8+ DC in the spleen during CMV infection (13).
In both humans and mice, subpopulations of NK cells show distinct in tissue distribution and expression of the lymph node (LN) homing receptor CD62L and chemokine receptors (14, 15, 16, 17, 18, 19, 20, 21, 22). Studies have shown that NK cells are present in resting (naive) LN at a very low frequency (∼0.5%), but that NK cells can enter into LN draining sites of immunization or infection where they may then influence the development of adaptive immunity (10, 19, 23, 24, 25). NK cells are recruited into draining LN after DC immunization and their subsequent IFN-γ production appears to be important for eventual CD4+ T cell Th1 differentiation (24). It has also been shown in mice that NK cells are important for inducing Th1 responses and early resistance to Leishmania major infection (23). A recent study using real-time imaging in vivo further revealed that NK cells localize near DC and interact with DC in resting LN, while additional NK cells are recruited into the draining LN after infection to produce key cytokines (25). Taken together, this evidence strongly suggests that an immune network among DC, T, and NK cells occurs within the local LN environment and determines the subsequent quality and type of Ag-specific immune response. Despite these advances, it has been unclear which NK cell subsets were recruited to the LN and what regulated this process and the level of NK cell priming. Mobilization of skin-resident DC has demonstrated two waves of migration into the draining LN. Dermal DC (DEC-205low) colonize the outer paracortex rapidly (within 24 h), while Langerhans DC colonize the inner paracortex more slowly (72 h) (26). NK cells are known to reside in the paracortex of LN (25), yet it is not yet known whether activated endogenous DC can activate NK cells in the draining LN.
In this study, we report that mature CD27high NK cells are recruited into the draining LN as a dominant population among distinct subtypes of NK cells. Such CD27high NK cell recruitment requires endogenous IFN-γ and is dependent upon the activation status of NK cells. Importantly, endogenous DC migration by hapten challenge also primes CD27high NK cell recruitment in an IFN-γ-dependent mechanism. Therefore, these results clearly indicate a critical contribution of endogenous IFN-γ in CD27high NK cell recruitment to the LN where DC migration and subsequent Ag presentation occurs.
Materials and Methods
Inbred wild-type (WT) C57BL/6 (B6) mice were purchased from the Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia). B6 IFN-γ−/− were bred and maintained at the Peter MacCallum Cancer Centre. All experiments were performed according to animal experimental ethics committee guidelines.
In vivo DC challenge
Bone marrow-derived DC were generated as previously described (27) from femurs of mice in the presence of cell culture supernatant from the GM-CSF- producing cell line and IL-4-producing cell line. DC maturation was induced by incubating the cells overnight with 2 μg/ml LPS (Sigma-Aldrich).
FITC labeling and DC enrichment
To induce migration of skin-resident DC, mice were painted on the ear with 12 μl of a 1% FITC (Sigma-Aldrich) solution prepared in 1:1 acetone:dibutlyphthalate. Following FITC painting, auricular LN were harvested at appropriate time points and subjected to gradient centrifugation to enrich DC. Briefly, LN were digested in collagenase (1 μg/ml) for 25 min at 37° C. Cells were then resuspended in 1.085 g/ml Optiprep and overlayed with 1.065 g/ml Optiprep before centrifugation at 600 × g for 15 min. DC were harvested from above the 1.065 g/ml solution while lymphocytes were harvested from the interface between the two solutions. Cells were then resuspended for analysis by flow cytometry.
For staining NK cells, mononuclear cells were first preincubated with CD16/32 (2.4G2) mAb to avoid the nonspecific binding of Abs to FcγR. Then the cells were incubated with a saturating amount of mAbs. Abs to CD3 (145-2C11), TCRβ (H57-597), NK1.1 (PK136), CD11b (M1/70), CD27 (LG.3A10), CD11c (N418), MHC II (M5/114.15.2), and CD205 (DEC-205) were purchased from BD Pharmingen, R&D Systems, eBioscience, or Miltenyi Biotec. Flow cytometric analysis was performed with an LSR II instrument (BD Biosciences). The number of NK cells was determined by the number of total mononuclear cells (trypan blue staining) and the proportion of NK cells (NK1.1+CD3− cells by flow cytometry) or the proportion of NK cell subsets (Mac-1lowCD27high, Mac-1highCD27high, and Mac-1high CD27low).
In vivo NK cell activation
Mice were injected i.p. with 2 μg of α-galactosylceramide (α-GalCer, provided by Pharmaceutical Research Laboratories, Kirin Brewery) or 200 μg of poly(I:C) (Sigma-Aldrich).
Data were analyzed for statistical significance using the Student t test. Values of p < 0.05 were considered significant.
Preferential recruitment of the mature CD27high NK cell subset to the DC-draining LN
It has been shown that NK cells are rapidly recruited to LN during immune responses and may play a supportive role in T cell priming (19, 24). As previously reported (24), we confirmed a rapid increase in the frequency and cellularity of NK cells within the DC-draining LN compared with control axial LN (Fig. 1, A and B). Even at the peak response (2 days), we did not detect any greater NK cell cycle activity within DC-draining LN compared with the control LN as determined by in vivo BrdU uptake (data not shown). Thus, in concert with previous observations (24), the increase in NK cell numbers was not due to cell proliferation.
In mice, the mature Mac-1high NK cell pool can be divided into two functionally distinct CD27high and CD27low subsets as distinct from phenotypically immature Mac-1low NK cells (16, 22). Aside from their differences in cytotoxic and cytokine effector functions, these NK cell subsets also show distinct migratory capacity and tissue distribution. To investigate which NK cell subpopulation was recruited to LN in vivo after immune challenge, we next determined the NK cell content of the draining LN at various times after DC challenge into the footpad. By dissecting NK cell subpopulations on the basis of Mac-1 and CD27 expression, we found that the mature Mac-1highCD27high (referred to as CD27high) NK cell subset was most effectively recruited into the DC draining LN. Interestingly, Mac-1lowCD27high (Mac-1low) NK cells persisted longer within the DC-draining LN after the initial peak response, whereas mature CD27high and CD27low Mac-1high NK cells were subsequently reduced in the DC-draining LN (Fig. 1, C–E). Among mature NK cell populations, the Mac-1highCD27low (CD27low) NK cell subset was a minor population within the DC-draining LN and naive LN (Fig. 1, C–E). These results indicated that, among NK cell subpopulations, the CD27high NK cell subset was preferentially recruited into DC-draining LN.
Contribution of host IFN-γ to optimal recruitment of NK cells to LN
Generally, lymphocyte recruitment is regulated by the interaction of chemokine-chemokine receptors and/or cell adhesion molecules, and CXCR3 is known to be a key chemokine receptor for NK cell LN recruitment (24). Among mature NK cells, the CD27high NK cell subset selectively expressed functional CXCR3 although there were no differences in CD62L expression (16). Interestingly, we observed that NK cells recruited into DC-draining LN down-regulate their CXCR3 expression at 16 h and further for 2 days after DC challenge compared with NK cells recruited into control LN (Fig. 2, A and B). In contrast to alteration in CXCR3 expression, we did not see any alteration in other cell surface molecule expression such as Ly49-C/I, Ly-49G2, or CD94 on LN-recruited NK cells (data not shown). These results suggest that the first wave of NK cell migration is CXCR3 dependent and further that CXCR3 ligation may then specifically down-regulate CXCR3 expression within DC-draining LN. To gain further insight into the molecular mechanisms contributing to NK cell recruitment into the DC-draining LN, we tested NK cell recruitment in gene-targeted mice responding to DC challenge. It is known that CXCR3 ligands are induced by IFN-γ and therefore we tested the importance of endogenous IFN-γ in NK cell recruitment into DC-draining LN. In IFN-γ−/− mice, we did observe a reduction in NK cell recruitment following footpad DC challenge (Fig. 2, C and D). Although the NK cells of IFN-γ−/− mice responded similarly to those in WT mice at early time points (16 h) after DC injection, there was no continued NK cell recruitment in IFN-γ−/− mice over the next 32 h to obtain a peak response 2 days after DC injection (Fig. 2, C and D). These results suggested that endogenous IFN-γ is key in obtaining an optimal NK cell recruitment into the DC-draining LN.
Endogenous DC migration induces recruitment of CD27high NK cells to draining LN in an IFN-γ-dependent mechanism
To further determine the physiological role of IFN-γ in the NK cell recruitment followed by DC migration into the LN, we analyzed DC migration and subsequent NK cell recruitment into DC-draining LN by using a contact hypersensitivity model. To induce epidermal DC migration to the draining LN, mice were painted with FITC dissolved within an irritant solution to induce migration and allow specific tracking of the migrated cells within the LN. Upon in vivo FITC challenge, both dermal (DEC-205lowFITC+) or Langerhans (DEC-205highFITC+) DC (gated within the CD11cintMHC IIhigh population) migrated into the draining LN with different kinetics (Fig. 3, A and B). NK cell recruitment was also primed following dermal DC migration and the CD27high subset was preferentially recruited after FITC challenge (Fig. 3, C and D). These results clearly indicate that endogenous DC migration induced by FITC challenge also primes CD27high NK cell recruitment into the draining LN.
We next determined the role of IFN-γ in NK cell recruitment following endogenous DC migration by examining NK cell recruitment in WT or IFN-γ−/− mice after FITC challenge. As shown in Fig. 4, the frequency and absolute number of NK cells in the DC-draining LN was significantly impaired in IFN-γ−/− mice compared with WT mice. Importantly, dermal DC migration in the IFN-γ−/− mice was not defective, but rather enhanced in the draining LN (data not shown) and, thus, the absence of NK cell recruitment in IFN-γ−/− mice is not simply due to impaired dermal DC migration/trafficking. Furthermore, IFN-γ−/− mice did not show any alteration in NK cell subpopulations in the DC-draining LN after FITC challenge (Fig. 4 C). Collectively, these results clearly indicate a critical role for endogenous host IFN-γ in NK cell recruitment upon endogenous DC migration induced by hapten challenging.
NK cells alter their migratory capacity upon activation
NK cells can be activated to produce cytokines and up-regulate their cytotoxic potential during immune responses. Both poly(I:C) and α-GalCer are known to potently activate NK cells through a TLR-dependent/type I IFN pathway (28) or invariant NKT cell-dependent/IFN-γ-mediated mechanism (29), respectively. To test whether the activation status of NK cells affected their migratory capacity to enter DC-draining LN, we tested the cellular composition of DC-draining LN in mice that had received α-GalCer (Fig. 5, A and C) or poly(I:C) (Fig. 5, B and D) 2 days before footpad DC challenge. Surprisingly, preactivated NK cells were not recruited into the DC-draining LN at either early (16 h) or late (2 days) phases of the response (Fig. 5, A–D). CXCR3 is known to be a key chemokine receptor for NK cell LN recruitment (24) and, among mature NK cells, the CD27high NK cell subset selectively expresses functional CXCR3 (16). Even though we have observed the selective expansion of CD27high NK cells upon in vivo NK cell activation using poly(I:C) or α-GalCer (30), these activated CD27high NK cells quickly down-regulated their CXCR3 expression compared with naive CD27high NK cells (Fig. 5 E). These results implied that activated NK cells may either be simply excluded from LN or unable to secondarily respond to DC challenge by simply down-regulating their key chemokine receptor CXCR3.
NK cells have been considered to be a potential initial source of IFN-γ for Th1 or CTL priming in the context of Ag-specific T cell responses (23, 24). In addition to predominantly localizing in the LN and showing a greater capacity to proliferate and produce cytokines (16), we have demonstrated that mature CD27high NK cells are the subset preferentially recruited to the draining LN following DC challenge. Importantly, we have also discovered that endogenous IFN-γ plays important role in NK cell recruitment into DC-draining LN. Finally, such an IFN-γ- dependent recruitment of CD27high NK cell subset is also observed when DC migration was induced by hapten challenge.
NK cells appear to be rapidly recruited to tissues at the site of infection or inflammation, and chemokines may play an important role in this process. Both human and mouse NK cells express a range of chemokine receptors that may attract them to different types of immune microenvironments (31, 32, 33). We demonstrated that the CD27high NK cell subset can be predominantly recruited into DC-draining LN upon DC challenge or hapten challenge. Several previous studies clearly showed that the broader NK cell pool can be recruited into LN during immune responses to provide an early IFN-γ (23, 24), and the chemokine receptor CXCR3 is known to play an important role in NK cell recruitment into the LN (24, 34). Given that the CD27high NK cell subset is the predominant resident mature NK cell population within secondary lymphoid organs in both mouse and humans (16, 35, 36) and further predominantly recruited into the draining site of the immune response, this subset may play a distinct role within LN during early priming phase of adaptive immune responses.
The importance of IFN-γ production by NK cells has been widely recognized as a direct effector mechanism. NK cell-derived early production of IFN-γ is also appreciated to play an important role in an effective priming of adaptive immune responses. Moreover, it has previously been shown that CXCR3 plays an important role in NK cell recruitment (24, 34) and CXCR3 ligands generally can be up-regulated by IFN-γ (37). Indeed, we have further demonstrated a critical contribution of IFN-γ in NK cell recruitment following either exogenous or endogenous DC migration into the LN. Since NK cells from IFN-γ−/− mice showed comparable expression of CXCR3 and chemotactic responsiveness to CXCR3 ligand in vitro (data not shown), the impaired NK cell recruitment to DC-draining LN in IFN-γ−/− mice was not simply due to a difference in CXCR3 sensitivity. We have demonstrated rapid down-regulation of CXCR3 expression on recruited NK cells into DC-draining LN and therefore the first wave of NK cell recruitment may be dependent on the chemotactic signal through CXCR3 and later migration may be amplified by endogenous IFN-γ production. Alternatively, CXCR3 down-regulation might be important to retain recruited NK cells within DC-draining LN or contribute to regulate NK cell egress from secondary lymphoid organs. Furthermore, we have previously reported that an altered NK cell-DC interaction occurs in the absence of IFN-γ−/− by a TNF-related apoptosis-inducing ligand-dependent mechanism (12), and it might be that this underlining mechanism potentially affects NK cell recruitment into secondary lymphoid organs. Because there was no defect in endogenous DC migration into the draining LN in IFN-γ−/− mice after hapten challenge (data not shown), the absence of NK cell recruitment in IFN-γ−/− mice following hapten-induced endogenous DC migration may not be simply due to impaired dermal DC migration. Surprisingly, prior NK cell activation by either poly(I:C) or α-GalCer, which is known to be type I and type II IFN dependent, respectively, diminished their recruitment to DC-draining LN. Upon in vivo NK cell activation using poly(I:C) or α-GalCer, we have observed the selective expansion of CD27high NK cells (30) and these activated NK cells quickly down-regulated their CXCR3 expression. Considering the importance of CXCR3 in NK cell recruitment to LN, activated NK cells may be excluded from LN by simply down-regulating their key chemokine receptor CXCR3. We postulate that this may be a safeguard mechanism to avoid an undesired contribution of preactivated NK cells during T cell priming. Alternatively, NK cells may have to be activated within the LN to be involved in T cell priming, since we observed that LN-recruited NK cells showed some signatures of activation following DC challenge, such as CXCR3 down-regulation. Interestingly, it has been shown that some, but not all, adjuvants induce NK cell recruitment into Ag-stimulated LN (24); however, the mechanism underlying what causes recruitment remains unclear. With our new data, the collective evidence might suggest that adjuvant activation of NK cells may be an important factor in altering the migration and recruitment of NK cells and hence their ability to prime T cells. In particular, we show that NK cell recruitment is tightly regulated by activation status and endogenous IFN-γ.
NK cells have been considered a “helper” population producing IFN-γ and capable of driving T cell priming without prior sensitization. Our present study has identified that mature CD27high NK cells preferentially recruited into DC-draining LN in an IFN-γ-dependent mechanism and may act as “accessory” cells during T cell priming. The mechanism by which IFN-γ controls NK cell recruitment to the draining LN remains to be established.
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.
Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; WT, wild type; MHC II, MHC class II; α-GalCer, α-galactosylceramide.