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* Division of Pediatric Rheumatology, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110;
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden;
University of California, San Francisco, CA 94143;
Genetics Institute, Wyeth Research, Cambridge, MA 02140;
¶ Department of Experimental Medicine and Pathology, University La Sapienza, Rome, Italy;
|| Centre d’Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique-Institut National de la Sante et de la Recherche Medicale-Universite de la Mediterranee, Marseille, France; and
# Howard Hughes Medical Institute and Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In addition to these effector functions, NK cells rapidly proliferate during viral infections, resulting in significantly expanded numbers of peripheral NK cells (15, 18, 19, 20, 21). This expansion occurs in two distinct phases, including an early nonspecific phase of NK cell proliferation, followed by a phase of preferential proliferation of NK cells that are able to recognize infected cells (15). The initial stage of nonspecific NK cell proliferation is presumably driven by cytokines induced early during viral infections in a similar manner to the nonspecific bystander proliferation observed in T cells during a viral infection or following poly(I:C) treatment (22, 23). The later specific phase is illustrated by the preferential proliferation of Ly49H+ NK cells during MCMV infection (15). Selective NK cell proliferation has also been observed during infection with the poxvirus vaccinia, demonstrating that this is not an MCMV- or even herpesvirus-restricted phenomenon (15). After resolution of the infection, the NK cell population contracts back to steady-state levels (20, 24).
The cytokines stimulating in vivo NK cell proliferation during infections are not well characterized, although there is evidence implicating the up-regulation of IL-15 by IFN-
in stimulating early, nonselective NK cell proliferation (19, 22, 25). However, efforts to directly delineate the contribution of IL-15 to viral-induced NK cell proliferation have been limited by the paucity of available reagents and the absence of NK cells in IL-15–/– and IL-15R–/– mice (26, 27). In addition, the complex interactions between IL-15 and its trimeric receptor have also hampered the investigation of the role of IL-15 in stimulating NK cell proliferation in vivo (28). For example, the coordinate expression of IL-15 and IL-15R and subsequent trans presentation to cells expressing the dimeric IL-2/15R common 
-chain receptor complex allow IL-15 stimulation to be tightly controlled and localized (29), but make quantification of IL-15 levels very difficult.
The factors driving the specific proliferation and expansion of the subset of NK cells that recognize infected cells during viral infections are even less well characterized and may involve cytokine stimulation and/or specific NK cell activation receptor-dependent signaling. For example, a recent report suggested that the specific expansion of splenic Ly49H+ NK cells during MCMV infection was IL-18 dependent (30). Previous work in our laboratory indicated that the selective proliferation of Ly49H+ NK cells during MCMV infection was dependent on Ly49H recognition of infected cells (15). Pretreatment of B6 mice with F(ab')2 of anti-Ly49H mAbs before MCMV infection resulted in significant reductions in overall NK cell proliferation and absolute NK cell numbers late during MCMV infection. Although this observation implicated Ly49H in contributing to selective NK cell proliferation, the anti-Ly49H treatment precluded the direct assessment of proliferation in the Ly49H+ and Ly49H– NK cell subsets due to residual bound F(ab')2. We have also recently reported that infection of B6 mice with m157-defective MCMV resulted in no accumulation of splenic Ly49H+ NK cells at day 4 or 5 postinfection (p.i.), providing indirect evidence that preferential proliferation is dependent on the interaction of Ly49H with m157 (31). However, neither of these studies conclusively demonstrated that the interaction between Ly49H and m157 was required for selective NK cell proliferation nor provided insight into how Ly49H-mediated recognition of infected cells resulted in preferential NK cell proliferation.
We hypothesized that Ly49H recognition of m157 on infected cells would cause differential responsiveness to cytokines other than those involved in stimulating early, nonspecific NK cell proliferation. This differential cytokine responsiveness could result from up-regulation of cytokine receptors on Ly49H+ NK cells that were absent on Ly49H– NK cells in an analogous manner to the up-regulation of IL-2Rs on T cells following engagement of the TCR (32). Alternatively, Ly49H activation could alter the response threshold for proproliferative cytokines, such as IL-15. We tested these hypotheses and found that Ly49H signaling mediated through DAP12 directly augments NK sensitivity to low concentrations of proproliferative cytokines, such as IL-15, resulting in preferential Ly49H+ NK cell proliferation.
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Materials and Methods |
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B6, C57BL/6.RAG1–/– (RAG–/–), C57BL/6.SCID (SCID), C57BL/6.IL-2–/– (IL-2–/–), C57BL/6.IL-4–/– (IL-4–/–), C57BL/6.IL-10–/– (IL-10–/–), C57BL/6.IL-12–/– (IL-12–/–), C57BL/6.IL-18–/– (IL-18–/–), and C57BL/6.IL-18R–/– (IL-18R–/–) mice were obtained from The Jackson Laboratory. B6 mice from National Cancer Institute (Charles River Laboratories) were also used. C57BL6.IL-15R–/– (IL-15R–/–) (26) mice were generated in the Ma laboratory (University of California). C57BL/6.DAP12 loss-of-function knock-in (DAP12KI) (33) mice were generated in the Vivier laboratory (Centre National de la Recherche Scientifique-Institut National de la Sante et de la Recherche Medicale-Universite de la Mediterranee). C57BL/6.IL-21R–/– (IL-21R–/–) (34) mice were provided by Wyeth-Ayerst Pharmaceuticals. Mice were maintained under specific pathogen-free conditions and used between 8 and 16 wk of age. All experiments were conducted in accordance with institutional guidelines for animal care and use.
IL-15R-deficient chimeric mice
IL-15R-deficient chimeric mice were generated, as previously described (35). Briefly, bone marrow was harvested from IL-15R–/– mice that had been backcrossed to B6 mice for at least eight generations or from wild-type (wt) B6 mice. Recipient B6 mice were irradiated with 950 rad total body irradiation, and then reconstituted with 3–6 x 106 bone marrow cells i.v. injected within 1 h of irradiation. NK cell chimerism was complete as determined by Ly-5.1 or IL-15R expression. Eight or more weeks after bone marrow transplant, the IL-15R-deficient chimeric mice as well as control chimeric mice reconstituted with wt bone marrow were infected with MCMV.
Abs and cell lines
The 3D10 Ab (anti-Ly49H) (36) was generated and biotinylated in our laboratory. PerCP-conjugated anti-CD3 (145-2C11), allophycocyanin-conjugated anti-NK1.1 (PK136), and FITC-conjugated anti-BrdU Abs as well as PE-streptavidin were all purchased from BD Pharmingen. Goat polyclonal, affinity-purified, biotinylated murine anti-IL-15R, anti-IL-17R, anti-IL-18R, and anti-IL-21R Abs were purchased from R&D Systems, and control polyclonal goat biotinylated Abs were purchased from Caltag Laboratories. The generation and characterization of BaF3 cells transfected with m157 (BaF3/m157) or with an empty vector (BaF3) have been previously described (13). RMA cells transfected with m157 (RMA/m157) were generated following the protocol used in the generation of BaF3/m157 transfectants (13). The generation and characterization of anti-m157 F(ab')2 and isotype control F(ab')2 have been previously described (37).
CFSE in vitro proliferation assays
Single-cell suspensions of splenocytes were prepared using standard techniques (15). NK cells from B6 and DAP12KI splenocytes were enriched from 
2.5 to 30–65% NK1.1+CD3– lymphocytes following negative selection with magnetic beads (Miltenyi Biotec NK cell negative selection kit). RAG–/– or SCID splenocytes (typically 30% NK1.1+CD3–) or enriched B6 or DAP12KI splenocytes were labeled with 2 µM CFSE (Molecular Probes) for 6 min at 37°C. The reaction was quenched by adding RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS (HyClone), and the cells were subsequently incubated for 30 min at 37°C per the manufacturer’s instructions. The CFSE-labeled splenocytes were plated in 96-well plates and cultured for 3 days in various concentrations of murine IL-15 (PeproTech) or low concentrations of IL-15 with either IL-2 (Chiron) or murine IL-18 (R&D Systems). BaF3 or BaF3/m157 cells were added to the wells over a range of E:T ratios (typically 3:1; 1.6:1; 0.8:1).
MCMV and infection of mice
Smith and K181 strains of MCMV were gifts from H. Virgin (Washington University, St. Louis, MO). A salivary gland stock of MCMV was prepared from young BALB/c mice that had been i.p. injected with 1 x 106 PFU tissue culture-propagated MCMV, and the titer of the stock was determined via standard plaque assay (6) using permissive NIH 3T12 fibroblasts (American Type Culture Collection). For in vivo experiments, mice were injected i.p. with 2 x 103 to 1 x 105 PFU/mouse salivary gland Smith MCMV stock or salivary gland K181 MCMV stock.
Splenocyte preparation, intracellular staining, and flow cytometry
To minimize the complication of BrdU+ NK cells from the bone marrow emigrating to the spleen, we used an acute pulse of BrdU (2 mg/mouse) injected i.p. 3 h before euthanizing the mice at various time points after infection rather than prolonged BrdU exposure in the drinking water. Single-cell suspensions of splenocytes were prepared using standard techniques (15). Splenocytes were incubated in 2.4G2 (anti-FcRII/III) supernatants (hybridoma from American Type Culture Collection) before staining with labeled Abs to block nonspecific binding of Abs to FcRs. Cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen), treated with DNase, and then stained with FITC-conjugated anti-BrdU Abs. Analysis was performed with a FACSCalibur flow cytometer (BD Pharmingen) gating on the NK1.1+CD3– lymphocyte subpopulation, and the data were analyzed with CellQuest (BD Pharmingen).
Selectivity index (SI)
An SI to assess the extent of preferential Ly49H+ NK cell proliferation was defined as the ratio of the percentage of Ly49H+ NK cells that were BrdU+ (fraction of overall NK cell population that was Ly49H+ and BrdU+ divided by the fraction of the overall NK cell population that was Ly49H+) compared with the percentage of Ly49H– NK cells that were BrdU+ (fraction of overall NK cell population that was Ly49H– and BrdU+ divided by the fraction of the overall NK cell population that was Ly49H–). An SI value of 1 denoted equal proliferation in Ly49H+ and Ly49H– NK cell subsets. An SI value greater than 1 represented preferential proliferation of Ly49H+ NK cells, and conversely an SI value less than 1 denoted less proliferation occurring in the Ly49H+ NK cell subset compared with the Ly49H– NK cell subset.
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Results |
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We previously observed two distinct phases of MCMV-induced NK cell proliferation with an early, nonspecific proliferative response, followed by preferential Ly49H+ NK cell proliferation in both the spleen and liver (15). To more carefully delineate the time course of these responses as well as to demonstrate their robustness and reproducibility, we analyzed acute BrdU incorporation in splenic NK cells from infected B6 mice in more than 35 independent experiments performed over a 3-year period (Fig. 1A). Splenic NK cells from naive mice were relatively quiescent undergoing only low levels of proliferation. Following MCMV infection, NK cell proliferation rapidly increased, consistently peaked at days 4–5 p.i. when 40% of the splenic NK cells incorporated BrdU over a 3-h period, and then waned after 6–7 days p.i. (Fig. 1A).
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Selective NK cell proliferation is mediated through DAP12 signaling
To test whether or not Ly49H signaling mediated via the ITAM-bearing adaptor DAP12 was necessary for stimulating viral-induced NK cell proliferation, we studied MCMV infection in loss-of-function DAP12KI mice. These mice express a signaling-deficient DAP12 molecule that facilitates relatively normal frequencies and surface expression levels of DAP12-dependent activation receptors, such as Ly49H, on NK cells when compared with wt mice (17, 33).
Following MCMV infection, the early nonspecific proliferative responses in the DAP12KI mice (15.0 ± 2.4% of NK cells were BrdU+) were similar to wt B6 mice (15.4 ± 2.7% of NK cells were BrdU+) with comparable increased BrdU incorporation in both the Ly49H+ and Ly49H– NK cell compartments, as illustrated by SI values of 0.97 ± 0.16 in DAP12KI mice (n = 5) and 1.03 ± 0.08 in B6 mice (n = 5) at day 1.5 p.i. (Fig. 2A). These data demonstrate that the nonspecific NK cell proliferative response observed early during MCMV infection is DAP12 independent.
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Ly49H signaling through DAP12 does not alter NK cell responsiveness to a panel of candidate cytokines
We hypothesized that Ly49H signaling through DAP12 would result in differential responsiveness to cytokines not involved in mediating early nonspecific NK cell proliferation, and that this differential cytokine responsiveness would lead to preferential Ly49H+ NK cell proliferation during MCMV infection. We tested this hypothesis by directly examining the surface expression of candidate cytokine receptors on splenic NK cells and the in vivo NK cell proliferative responses in cytokine and cytokine receptor knockout mice during MCMV infection. We focused on cytokines, such as IL-2, IL-12, and IL-18, which have been shown to stimulate in vitro NK cell responses as well as a number of T cell-derived cytokines, including IL-4, IL-10, IL-17, and IL-21.
Expression of candidate cytokine receptors (common -chain (IL-2/15R), IL-2/15R, IL-2R, IL-4R, IL-12R, IL-17R, IL-18R, and IL-21R) was evaluated on splenic NK cells from naive and infected (day 4 p.i.) B6 mice. Although several cytokine receptors were slightly up-regulated following MCMV infection, including the common -chain, IL-12R, IL-18R, and IL-21R, only the surface expression of IL-2R (and IL-15R; see below) was significantly up-regulated (Fig. 3A). In addition, there was no differential expression of any of these receptors (including IL-2R) on Ly49H+ or Ly49H– NK cell subsets following MCMV infection (with the exception of very subtle changes in IL-4R and IL-18R expression on Ly49H– NK cells) (Fig. 3B). Thus, differential expression of these candidate cytokine receptors does not account for the selective Ly49H+ NK cell proliferation observed during MCMV infections.
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Our results with IL-18–/– and IL-18R–/– mice conflict with a previous report implicating IL-18 in the specific expansion of Ly49H+ NK cells during MCMV infection (30). Due to this discrepancy, we examined NK cell proliferation in IL-18–/– and IL-18R–/– mice in greater detail and found no significant differences in overall NK cell proliferation over the course of MCMV infection in either IL-18–/– (Fig. 4A) or IL-18R–/– (Fig. 4B) mice compared with IL-18+/– littermates and/or wt B6 mice. Furthermore, we found no deficits in the selective expansion and accumulation of splenic Ly49H+ NK cells in IL-18–/– (Fig. 4C) or IL-18R–/– (Fig. 4D) mice following MCMV infection. The extent of the selective expansion of splenic Ly49H+ NK cells is dependent on the inoculum dose of MCMV; however, we observed no differences in splenic Ly49H+ NK cell expansion and accumulation in IL-18–/– mice compared with IL-18+/– littermates or wt B6 mice following infection over a range of lower inoculum doses (2 x 103 – 1 x 104 PFU/mouse; data not shown) comparable to those used by Andrews et al. (30). The absence of proliferation defects in IL-18–/– and IL-18R–/– mice demonstrates that IL-18 either does not participate or plays a noncritical, redundant role in the specific proliferation and subsequent accumulation of Ly49H+ NK cells during infection with Smith strain MCMV.
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on NK cells in modulating selective proliferation
IL-15 is crucial for NK cell development, homeostasis, and survival, and has been implicated in stimulating NK cell proliferation during viral infections (19). Given the inherent difficulties in blocking IL-15 in vivo, we focused our investigations on the role of IL-15R in viral-driven, selective NK cell proliferation. Because IL-15R–/– mice lack NK cells (26), we used IL-15R-deficient chimeric mice in which bone marrow from IL-15R-deficient mice was used to reconstitute irradiated wt recipients. In these chimeric mice, host cells expressing IL-15R efficiently present IL-15 in trans to IL-15R-null NK cells promoting their development and survival (29, 35).
In contrast to most of the cytokine receptors that we have examined, IL-15R is up-regulated on splenic NK cells from wt mice following MCMV infection (day 4 p.i.), as evidenced by the increase in mean fluorescence intensity of IL-15R from 26 on NK cells from naive wt mice to 131 on NK cells from MCMV-infected wt mice (Fig. 5A). However, as was seen with IL-2R, no differential expression of IL-15R was observed on Ly49H+ compared with Ly49H– NK cells from infected mice (data not shown). The up-regulation of IL-15R following MCMV infection could also be observed on NK cells from wt chimeric mice (generated by reconstituting irradiated wt recipients with wt bone marrow; Fig. 5A).
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on NK cells during MCMV infection could enhance NK cell proliferation by facilitating cis presentation of IL-15 on NK cells potentially prolonging IL-15 stimulation. However, no deficits were observed in NK cell proliferation (Fig. 5B) or the expansion of Ly49H+ NK cells (Fig. 5C) in the IL-15R-deficient chimeric mice compared with the wt chimeric mice on day 4 or 5 p.i. with MCMV. The SI values of 1.8 ± 0.14 and 2.2 ± 0.5 in the IL-15R-deficient chimeric mice on day 4 or 5 p.i. demonstrate that selective Ly49H+ NK cell proliferation was not compromised by the absence of IL-15R on NK cells (Fig. 5C). Therefore, up-regulation of IL-15R on wt NK cells did not facilitate selective NK cell proliferation during MCMV infection. m157 recognition in the context of low concentrations of proproliferative cytokines is sufficient to stimulate selective NK cell proliferation
Although we observed no evidence that up-regulation of IL-15R on NK cells contributed to preferential NK cell proliferation, the possibility remained that Ly49H stimulation could directly enhance IL-15-dependent NK cell proliferation. We tested this hypothesis in vitro by incubating enriched, CFSE-labeled B6 NK cells with target cells in varying concentrations of IL-15. Low IL-15 concentrations stimulated preferential proliferation of Ly49H+ NK cells as measured by CSFE dilution when the NK cells were incubated with BaF3 cells transfected with m157, but no preferential proliferation when the NK cells were incubated with BaF3 targets lacking m157 expression (Fig. 6A). Incubation of NK cells without targets in the same low concentrations of IL-15 promoted only low levels of NK cell proliferation. At low IL-15 concentrations, we consistently saw a small decrease in NK cell proliferation with the addition of target cells presumably reflecting competition for cytokines or other components in the medium, but this did not obscure the differences between Ly49H+ and Ly49H– NK cell proliferation in the presence of BaF3/m157 targets. Preferential proliferation of Ly49H+ NK cells was also observed when CFSE-labeled B6 NK cells were incubated in low IL-15 concentrations with syngeneic RMA cells transfected with m157, but not with RMA cells lacking m157 expression (Fig. 6B). As IL-15 concentrations were increased, broad-based, nonspecific stimulation of NK cell proliferation was observed regardless of Ly49H expression or the presence of m157 on the targets (Fig. 6A). Preferential proliferation of Ly49H+ NK cells was abrogated in the presence of F(ab')2 of anti-m157 Abs, demonstrating the specificity and nonredundancy of the interaction between Ly49H and m157 in stimulating preferential NK cell proliferation (Fig. 6C). Together with previous studies demonstrating that m157 is only recognized by Ly49H and not other NK cell receptors on B6 NK cells (12, 13), these results demonstrate that the interaction between Ly49H and m157 in the appropriate cytokine milieu is sufficient to mediate selective NK cell proliferation.
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Ly49H signaling through DAP12 directly augments stimulation by proproliferative cytokines
To verify the role of DAP12 in mediating Ly49H stimulation of preferential NK cell proliferation, the experiments described above were repeated with enriched splenic B6 and DAP12KI NK cells in parallel. When B6 and DAP12KI splenocytes were incubated in low IL-15 concentrations with BaF3/m157 targets, the B6 splenocytes underwent preferential proliferation of Ly49H+ NK cells, whereas no preferential NK cell proliferation was observed in the DAP12KI splenocytes (Fig. 6E). This verifies the nonredundant role of DAP12 in Ly49H-mediated stimulation of selective NK cell proliferation. The DAP12KI NK cells were not deficient in their ability to proliferate in response to IL-15 stimulation, as demonstrated by the nearly identical proliferative responses of both DAP12KI and B6 NK cells at higher IL-15 concentrations (Fig. 6F). Although Ly49H signaling mediated through DAP12 was necessary for selective NK cell proliferation, DAP12 signaling in the absence of proproliferative cytokine stimulation was not sufficient to stimulate NK cell proliferation or even to support NK cell survival, as demonstrated by the incubation of B6 NK cells with BaF3/m157 targets in the absence of IL-15 for 3 days (data not shown) or by the lack of preferential NK cell proliferation observed in the coculture of B6 NK cells and BaF3/m157 targets in the presence of very low concentrations of IL-15, which alone did not stimulate NK cell proliferation (Fig. 6D). Taken together, these results illustrate that the interaction between Ly49H and m157 results in signaling mediated via DAP12 that amplifies the response of Ly49H+ NK cells to stimulation by low concentrations of proproliferative cytokines such as IL-15.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We found no evidence that Ly49H signaling via DAP12 resulted in the selective up-regulation of cytokine receptors or increased responsiveness to cytokines that are different from those involved in stimulating nonspecific NK cell proliferation. Several candidate cytokine receptors, including IL-2R and IL-15R, were strongly up-regulated on NK cells during MCMV infection. However, our studies detected no deficits in NK cell proliferation or Ly49H+ NK cell accumulation in IL-15R-deficient chimeric mice compared with wt chimeric mice during MCMV infection, indicating that cis presentation of IL-15 by IL-15R on NK cells is not crucial to MCMV-induced selective NK cell proliferation. In addition, the lack of differential expression of IL-2R on Ly49H+ and Ly49H– NK cells, the normal preferential Ly49H+ NK cell proliferation observed in IL-2–/– mice, and the normal Ly49H+ NK cell expansion and accumulation in IL-2–/– mice during MCMV infection (data not shown) demonstrate that IL-2 and IL-2R do not play critical, nonredundant roles in mediating preferential NK cell proliferation during MCMV infection. We have observed subtle, but reproducible decreases in the extent of preferential proliferation of splenic Ly49H+ NK cells in RAG–/– mice (A. French, unpublished data), raising the possibility that a different T cell-derived factor, such as IL-21, could play a minor, nonredundant role in this process. However, we found no deficits in preferential NK cell proliferation or Ly49H+ NK cell expansion in IL-21R–/– mice following MCMV infection. In addition, we found no evidence that three other T cell-derived cytokines (IL-4, IL-10, and IL-17) play a role in MCMV-induced NK cell proliferation.
A previous report noted that Ly49H+ NK cell accumulation was deficient in IL-18–/– mice and concluded that IL-18 may play a role in supporting the selective expansion of Ly49H+ NK cells during MCMV infection (30). It is difficult to reconcile this conclusion with our results. One potential explanation is that our experiments were performed with Smith strain MCMV, while the experiments of Andrews et al. (30) were performed with K181 Perth strain MCMV. However, we observed similar results when we repeated the experiments with 104 PFU/mouse K181 strain MCMV (data not shown), although it is possible that differences exist in the Perth strain K181 used in Andrews’ study and the K181 strain that we used. Another potential explanation lies in the completeness of the backcrossing of the IL-18–/– mice. For example, the DAP12 locus in the Australian IL-18–/– mice may remain from the 129 parental strain, resulting in a dysfunctional molecule that does not effectively transmit Ly49H signals (41). Alternatively, one could argue that we saw normal Ly49H+ NK cell expansion in our IL-18–/– mice because they were not true knockouts. However, based on PCR genotyping, RNA protection assay for IL-18 RNA following MCMV infection, and decreased IFN- production following MCMV infection (data not shown), our IL-18–/– mice were IL-18 deficient and still manifested normal preferential NK cell proliferation and Ly49H+ NK cell accumulation following infection with Smith strain MCMV over a wide range of MCMV inoculum doses. Furthermore, our data with IL-18–/– mice were identical with results in IL-18R–/– mice strongly supporting the conclusion that IL-18 plays a dispensable or redundant role in stimulating NK cell proliferation during MCMV infection.
Our findings indicate that Ly49H signaling mediated via DAP12 amplifies the response of Ly49H+ NK cells to low concentrations of proproliferative cytokines. The question of how DAP12 signaling augments proproliferative cytokine stimulation is currently under investigation. It is possible that DAP12 signaling directly influences proproliferative cytokine stimulation by enhancing JAK/STAT phosphorylation or by suppressing SOCS (suppressor of cytokine signaling) proteins. Recent work by Ortaldo et al. (42) demonstrated that ITAM-mediated signaling in NK cells enhanced IL-12-stimulated IFN- production through modulation of p38 MAPK- and ERK-dependent signaling pathways; however, further study is needed to determine the intermediaries in the cross-talk between DAP12 and proproliferative cytokine signaling.
Our observations support a model in which viral-induced, selective NK cell proliferation is a function of both proproliferative cytokine concentrations and the effective activation receptor ligand "concentration" in the NK cell environment. The interplay between these two variables dictates the observed NK cell proliferative response, as illustrated in the case of MCMV infection. In the absence of m157, low IL-15 concentrations promote the survival of NK cells without stimulating significant proliferation. When proproliferative cytokine concentrations are high, such as early during viral infections or following poly(I:C) injection, NK cells are stimulated to proliferate regardless of Ly49H expression (poly(I:C); A. French, unpublished results). Under conditions of high proproliferative cytokine concentrations, m157 stimulation of Ly49H+ NK cells is effectively masked by the strong cytokine-mediated proliferative stimulus to all NK cells (although it is possible that early during infection, low m157 expression also contributes to the lack of specific Ly49H+ NK cell proliferation). As the proproliferative cytokine concentrations fall and/or the effective concentration of m157 increases, the impact of Ly49H recognition of m157 and subsequent signaling via DAP12 becomes manifest in preferential proliferation of Ly49H+ NK cells. Ly49H signaling appears to alter the response threshold of NK cells, allowing them to respond to lower concentrations of proproliferative cytokines than unstimulated NK cells. Therefore, the impact of m157 will be most significant as the proproliferative cytokine concentrations fall to levels that alone provide little proliferative stimulus. Eventually, the NK cell population contracts presumably as m157 (i.e., MCMV infection) is cleared and proproliferative cytokine levels wane, resulting in steady-state IL-15 concentrations that are not able to support the expanded numbers of NK cells.
In many ways, the viral-induced preferential proliferation of Ly49H+ NK cells resembles the clonal expansion seen in T cells following infections. Although NK cell receptors like Ly49H are not clonally restricted, they associate with ITAM-containing signaling molecules and use Syk family tyrosine kinase activation pathways in a similar manner to TCRs. In the appropriate cytokine milieu, specific receptor stimulation of NK cell receptors as well as TCRs results in preferential proliferation and selective expansion of the receptor-specific NK or T cells. Although the specific proliferation of Ly49H+ NK cells leads to relatively prolonged (i.e., 4-wk) perturbations in splenic NK cell populations (A. French, unpublished observation), it remains to be determined whether this selective expansion of virus-specific NK cells results in functional immunological memory with a more vigorous NK cell response upon re-exposure to the infective stimulus in an analogous manner to T cells.
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This research was supported by a National Institute of Allergy and Infectious Diseases K08 Grant and a Howard Hughes Medical Institute Faculty Development Award (to A.R.F.); Barnes-Jewish Hospital Research Foundation and National Institutes of Health grants to W.M.Y., who is a Howard Hughes Medical Institute investigator; and grants from the Erik and Edith Fernströms Stiftelse för Medicinsk Forskning and the Swedish Research Council (to K.K. and H.S.). 
2 Address correspondence and reprint requests to Dr. Anthony R. French, Washington University, Box 8208, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: french_a{at}kids.wustl.edu
3 Abbreviations used in this paper: MCMV, murine CMV; DAP12, DNAX-activation protein 12; p.i., postinfection; SI, selectivity index; wt, wild type.
Received for publication March 27, 2006. Accepted for publication July 17, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, IL-12, and IL-15 regulation of NK cell responses to viral infection. J. Immunol. 169: 4279-4287. recycles and presents IL-15 in trans to neighboring cells. Immunity 17: 537-547. [Medline] and IL-15 supports natural killer cell and memory CD8+ T cell homeostasis. J. Exp. Med. 200: 825-834. -deficient natural killer cells survive in normal but not IL-15R-deficient mice. J. Exp. Med. 197: 977-984. 2-microglobulin-deficient mice. J. Immunol. 169: 126-136. This article has been cited by other articles:
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  T. L. Geurs, Y. M. Zhao, E. B. Hill, and A. R. French Ly49H Engagement Compensates for the Absence of Type I Interferon Signaling in Stimulating NK Cell Proliferation During Murine Cytomegalovirus Infection J. Immunol., November 1, 2009; 183(9): 5830 - 5836. [Abstract] [Full Text] [PDF]
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 S.-H. Lee, K.-S. Kim, N. Fodil-Cornu, S. M. Vidal, and C. A. Biron Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection J. Exp. Med., September 28, 2009; 206(10): 2235 - 2251. [Abstract] [Full Text] [PDF]
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J. C. Sun, A. Ma, and L. L. Lanier Cutting Edge: IL-15-Independent NK Cell Response to Mouse Cytomegalovirus Infection J. Immunol., September 1, 2009; 183(5): 2911 - 2914. [Abstract] [Full Text] [PDF]
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M. T. Orr, J. C. Sun, D. G.T. Hesslein, H. Arase, J. H. Phillips, T. Takai, and L. L. Lanier Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection J. Exp. Med., April 13, 2009; 206(4): 807 - 817. [Abstract] [Full Text] [PDF]
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 X. Chen, F. Bai, L. Sokol, J. Zhou, A. Ren, J. S. Painter, J. Liu, D. A. Sallman, Y. A. Chen, J. A. Yoder, et al. A critical role for DAP10 and DAP12 in CD8+ T cell-mediated tissue damage in large granular lymphocyte leukemia Blood, April 2, 2009; 113(14): 3226 - 3234. [Abstract] [Full Text] [PDF]
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N. Zucchini, G. Bessou, S. Traub, S. H. Robbins, S. Uematsu, S. Akira, L. Alexopoulou, and M. Dalod Cutting Edge: Overlapping Functions of TLR7 and TLR9 for Innate Defense against a Herpesvirus Infection J. Immunol., May 1, 2008; 180(9): 5799 - 5803. [Abstract] [Full Text] [PDF]
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M. Divangahi, T. Yang, K. Kugathasan, S. McCormick, S. Takenaka, G. Gaschler, A. Ashkar, M. Stampfli, J. Gauldie, J. Bramson, et al. Critical Negative Regulation of Type 1 T Cell Immunity and Immunopathology by Signaling Adaptor DAP12 during Intracellular Infection J. Immunol., September 15, 2007; 179(6): 4015 - 4026. [Abstract] [Full Text] [PDF]
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) and DAP12KI (
) splenic NK cells at 1.5, 2, and 3 days p.i. with MCMV (5 x 104 PFU/mouse) with four to five mice per group. B, Proliferation of splenic Ly49H+ and Ly49H– NK cells was assessed by intracellular staining for acute BrdU incorporation on day 2 p.i. with 5 x 104 PFU/mouse MCMV in wt and DAP12KI mice. Flow cytometry analysis was gated on NK1.1+CD3– splenic lymphocytes. The percentage of NK cells in each quadrant is denoted in the upper right-hand corner of the scatterplots. Plots are representative of four individual mice within each group.
, represent IL-18R–/– mice (four mice per group). Note wt B6 cohort at day 4 p.i. in A and B are from the same experiment. C, Percentage of splenic NK cells (NK1.1+CD3–) that are Ly49H+ assessed by flow cytometry at various days during the course of MCMV infection (5 x 104 PFU/mouse) in IL-18–/– mice. 
