Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: J Immunol. 2014 Jun 6;193(1):354–363. doi: 10.4049/jimmunol.1302999

Proliferation Conditions Promote Intrinsic Changes in NK Cells For an IL-10 Response

Margarite L Tarrio *,1, Seung-Hwan Lee *,1,2, Maria F Fragoso *, Hong-Wei Sun †, Yuka Kanno ‡, John J O’Shea ‡, Christine A Biron *
PMCID: PMC4065839  NIHMSID: NIHMS592271  PMID: 24907347

Abstract

Constitutively found at high frequencies, the role for NK cell proliferation remains unclear. Here, a shift in NK cell function from predominantly producing interferon-γ (IFN-γ), a cytokine with proinflammatory and antimicrobial functions, to producing the immunoregulatory cytokine IL-10, was defined during extended murine cytomegalovirus infection. The response occurred at times subsequent to IL-12 production, but the NK cells elicited acquired responsiveness to IL-12 and IL-21 for IL-10 production. Because neither IL-12 or IL-21 was required, however, additional pathways appeared to be available to promote the NK cell IL-10 response in vivo. In vitro studies with IL-2 to support proliferation and in vivo adoptive transfers into murine cytomegalovirus-infected mice demonstrated that NK cell proliferation and further division enhanced the change. In contrast to the sustained open profile of the IFN-γ gene, NK cells responding to infection acquired histone modifications in the IL-10 gene indicative of changing from a closed to an open state. The IL-10 response to IL-12 was proliferation dependent ex vivo if the NK cells had not yet expanded in vivo but independent if they had. Thus, a novel role for proliferation in supporting changing innate cell function is reported.

Keywords: Natural Killer Cells, IL-10, IFN, IL-12, Murine Cytomegalovirus

Introduction

Natural killer (NK) cells of the innate immune system are important in early defense against viral infections of mice and humans. In addition to being induced to mediate direct antiviral effects, they are stimulated by cytokines and through activating receptors to undergo proliferation (1–5). The biological importance of this response has remained elusive. In contrast to the low numbers of antigen-specific T cell subsets needing expansion for defense, NK cells are basally found at high frequencies and large proportions express particular activating receptors (5). During early murine cytomegalovirus (MCMV)4 infection, induced IL-12 stimulates NK cell production of IFN-γ to support proinflammatory effects and consequently, enhance innate antiviral defense (3, 6). NK cells can sometimes make the immunoregulatory cytokine IL-10 (4, 7–12), and they do so at later times during sustained MCMV replication as a result of deficiency in cytotoxic function (4). Under these conditions, NK cell IL-10 acts to limit adaptive responses for protection against immune-mediated disease.

So then, how are NK cells regulated to balance their proinflammatory and immunoregulatory effects? Extrinsic factors such as the cytokines available to stimulate IL-10 expression might be differentially induced to support these responses. IL-12 can sometimes elicit, and other cytokines can conditionally induce, IL-10 (7, 9, 13–17), but their roles in eliciting NK cell IL-10 during viral infection have not been examined. Alternatively, there could be intrinsic changes in cellular “preparedness” to make IL-10 (18, 19). Remarkably, the NK cell IL-10 response during sustained MCMV replication in cytotoxic-deficient mice is observed subsequent to their proliferation (4). Because these observations suggest that the events may be linked, experiments were carried out characterizing conditions of MCMV infections in immunocompetent wild type (Wt) mice associated with an NK cell IL-10 response. The results show that extrinsic factors can play a role. During extended infection, however, NK cells are intrinsically changed to access an IL-10 response with histone modifications for opening up the IL-10 gene. The altered function is dependent on proliferation competence and/or previous division. Thus, a new role for proliferation in supporting changing function of an innate cell response is reported, and one answer to the question of why NK cells proliferate is provided.

Materials and Methods

Mice

Mice were on B6 background. The CD45.2 C57BL/6 and CD45.1 allotypic mice were purchased from Taconic Farms (Germantown, NY). Breeding pairs of B6 mice with the “knock-in” insertion of a genetic construct with an internal ribosomal entry site and the GFP gene under the transcriptional control of the IL-10 gene (interleukin-ten ires gfp-enhanced reporter, Tiger) (20) i.e. IL-10-GFP-reporter, and IL-12p35-deficient (21) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Breeding pairs of IL-21- and IL-21 receptor-deficient mice (22) were from M. Ricon (University of Vermont). The CD45.1 Ly49H-deficient (4, 23) mice were bred. Only IL-10-reporter mice heterozygous for the GFP containing knock-in allele were used for all studies. The IL-10-GFP reporter allele was introduced into IL-21-deficient and IL-21 receptor-deficient by crossing with IL-10-GFP reporter mice. Mice were used at 6-to-14 wks of age. Animals obtained from sources outside of Brown University were housed in the animal care facility for at least one week before use. Handling of mice and experimental procedures were conducted in accordance with institutional guidelines for animal care and use, and protocols were approved by the Brown University Institutional Animal Care and Use Committee (IACUC).

In Vivo Manipulation

Except where indicated, infections were i.p. with 4,000/5,000 (low dose) or 15,000/70,000 (high dose) plaque forming units (PFUs) of salivary gland-derived Smith MCMV derived from ATCC as described previously (4). For IL-12/23 blocking, 750 ug of an α-IL-12p40 (the clone C17.8 anti-mouse IL-12/23; BioXCell) was administered by i.p. injection at 4 h prior to infection. For controls, an equal amount of rat IgG2a (BioXCell) was given.

Sample Preparations and Assays

Mice were anesthetized using Isoflurane (Aerrane, Baxter Healthcare), and blood was collected via the retro-orbital route into tubes containing heparin. Samples were centrifuged at 10,000 rpm for 10 min. Supernatants were identified as sera because there was always some clotting. The viral titers in homogenized organ samples were quantified by standard plaque assays using mouse embryonic fibroblasts (4). Cytokine levels were measured and are presented as pg/ml for serum or pg/106 cells for conditioned media: IFN-α and IFN-β by ELISA (R&D Systems); IL-21 by ELISA (MBL International); IFN-γ, IL-12p70, IL-6, and IL-10 using BD Cytometric Bead Assays (CBA). For RT-PCR, primers synthesized by Operon were: IL-10 (forward: 5’-TGCTATGCTGCCTGCTCTTACTGA-3’; reverse: 5’-CCTGCTCCACTGCCTTGCTCTTAT-3’) (14), IL-21 (forward: 5’- CCCTTGTCTGTCTGGTAGTCATC-3’; reverse: 5’-ATCACAGGAAGGGCATTTAGC-3’) (24), and Gapdh (forward, 5’-ACCACAGTCCATGCCATCAC-3’; reverse, 5’-TCCACCACCCTGTTGCTGTA-3’). Cell and mRNA samples were from spleens.

Flow Cytometric Analyses

Splenic leukocytes were incubated for 20 min with 2.4G2 antibody to reduce nonspecific staining. Cell surface markers were identified using labeled antibodies (BD Biosciences and eBioscience): NKp46-phycoerythrin (PE), TCR-β-fluorescein (FITC), TCR-β-PE, TCR-β-Peridinin-Chlorophyll-Protein-Complex-Cy5.5 (PerCP5.5), TCR-β-allophycocyanin (APC), TCR-β-allophycocyanin-Cy7 (APC-Cy7), TCR-β-phycoerythrin-Cy7 (PE-Cy7), Ly49H-APC, NK1.1-PE, NK1.1-Peridinin-Chlorophyll-Protein-Complex (PerCP), NK1.1-APC, CD45.2- PerCP5.5, CD45.2-FITC, CD49b-FITC, CD49b-PE-Cy7, CD49b-APC, and CD49b-PE. For intracellular IFN-γ, cells were brefeldin A treated (5 ug/ml) for 4 h, surface stained, fixed/permeabilized (in CytoFix/CytoPerm buffer; BD Biosciences), and labeled with anti-IFN-γ-PE. For increased GFP detection, a recombinant rabbit monoclonal anti-GFP antibody (Invitrogen), followed with an Alexa Fluor 488-labeled goat anti-rabbit IgG (Invitrogen), were added to fixed/permeabilized cells. Samples were acquired using either a FACSCalibur or FACSAria (BD Bioscience), and data were analyzed with either the CellQuest Pro (BD Bioscience) or FlowJo (Tree Star, Inc) software. The FlowJo Proliferation platform was used for analysis of cell division.

NK Cell Preparations and Ex Vivo Manipulations

For conditioned media, 5×105 total or 1×105 purified cells were incubated in the presence or absence of mouse recombinant cytokines, IL-12 (10 or 50 ng/ml, eBioscience), IL-21 (100 ng/ml, eBioscience), IL-22 (100 ng/ml, Peprotech), IL-23 (100 ng/ml, R&D Systems), or IL-27 (100 ng/ml, R&D Systems). Supernatants were harvest after 24 h. Splenic NK and T cell subsets were FACS sorted (FACSAria; Flow Cytometer Facility at Brown University) based on the expression of CD49b and TCR-β using leukocytes from day 3.5 infected mice stained with PE-CD49b and APC-TCR-β. Purity was >80 to 92%. Where indicated, NK cells were prepared, using immune-magnetic bead selection (Stem Cell Technologies Negative Selection Mouse NK Cell Enrichment Kit), to a purity of ~80%, i.e. 76-to-85% on d 0, and 85-to-87% on d 3.5, with 40% as compared to 60% of the NK cells expressing Ly49H in d 0 as compared to d 3.5 preparations. Thus, their frequencies in the populations were increased from 4 to 6 out of 10. The cells were labeled with 5µM Cell Proliferation Dye eFluor670 (eBioscience), washed and used in culture or in adoptive transfers. In some experiments, cells were pre-treated with 50µg/ml Mitomycin C (Sigma-Aldrich) to block proliferation. Splenic leukocytes (1×106) or purified NK cells (1×105) were cultured for 48 or 72 h in the presence of 25 or 1000 U/ml IL-2 (eBioscience, sp. act. ≥5.7×106 U/mg), with or without the addition of 50 ng/mL IL-12 (eBioscience, ≥5.7×106 U/mg) 24 h prior to harvest.

Adoptive Transfer

For adoptive transfer experiments, 2×107 splenic leukocytes or 4×106 purified NK cells from CD45.2 IL-10-GFP reporter mice, that were uninfected or infected with 70,000 PFU MCMV for the indicated times, were adoptively transferred by i.v. injection to either Wt or Ly49H−/− B6 mice with the CD45.1 allotypic marker. Where indicated, the cells were labeled with eFluor670 Proliferation Dye (eBioscience) before transfer. The Wt recipient mice were infected with 5,000 PFU and the Ly49H−/− mice were infected with 4,000 PFU of MCMV as described. Approximately 1.5 to 3.5 millions of cells were acquired per sample and CD45.2 donor NK and T cells were analyzed for IL-10-GFP and IFN-γ expression.

Chromatin Immunoprecipitation Assays

NK cells were purified from uninfected mice and MCMV- infected mice at day 1.5 and day 3.5 by negative selection. Chromatin immunoprecipitation was carried out using antibodies against H3K4 trimethylation (Millipore #04–745), H3K27 trimethylation (Millipore #07–449) and H3K36 trimethylation (AbCam #9050). Recovered DNA of approximately 10 to 50 ng were made into libraries with illumine adaptors and sequenced for single read 50 cycles using HiSeq 2000 (Illumina, San Diego, CA). Uniquely matching reads are mapped to mouse mm9 reference genome and significant islands were called with SICER (25). Integrative Genomics Viewer (Broad Institute) was used to generate histone mark distribution around the IL-10 locus. The data have been added to the GEO repository (GSE55834).

Statistical Analysis

Statistical significance of differences was determined by the unpaired two-tailed student’s t test. Where indicated, significance of differences between multiple groups was also evaluated by ANOVA.

Results

IL-10 production by NK cells during sustained viral infection

Following low-dose MCMV infection of Wt B6 mice with 5,000 plaque forming units (PFU), virus was controlled in spleens by d 2 but prolonged in livers (Fig. 1A). The expected (3, 6, 26–28) systemic innate cytokine IFN-α, IL-12p70, IL-6, and IFN-γ responses peaked on d 1.5 of infection with IFN-β levels below detection (Fig. 1B). Serum IL-10 was marginally detectable in a subset of samples at later times. High-dose infection with 70,000 PFU resulted in sustained and elevated viral replication in spleens and livers, with similar kinetics of early serum cytokine responses now having higher magnitudes and detectable IFN-β. Notably, IL-10 was elicited but temporally distinct from the other cytokine responses, with increases on d 3.5. Splenic IL-10 mRNA elevation started earlier and reached higher levels during high-dose infection (Fig. 1C). Serum IL-21, another cytokine reported to induce IL-10, was only found in a few samples from high-dose-infected mice (Fig. 1D), but either dose stimulated IL-21 mRNA expression (Fig. 1E).

FIGURE 1. Viral replication and cytokine production during MCMV infection at low and high doses.

FIGURE 1

Open in a new tab

Mice, Wt B6, were infected with 5,000 (low dose) or 70,000 (high dose) PFU MCMV for indicated times. (A) Spleen and liver virus was determined by plaque assay. (B) Serum levels of IFN-α and IFN-β were measured by ELISA, and of IL-12p70, IL-6, IFN-γ, and IL-10 by CBA. For A and B, 5,000 PFU data (mean ± s.d. of 3 mice) are representative of two, and 70,000 PFU data (mean ± s.d. of 3 mice) of three, experiments. (C) Splenic Il-10 mRNA was measured by semi-quantitative RT-PCR with Gapdh mRNA used as input control. Results are representative of three experiments. (D) Serum IL-21 levels were measure using ELISA. Each symbol represents an individual mouse. Bars are averages. (E) Splenic Il-21 mRNA was measured by semi-quantitative RT-PCR with Gapdh as control. Results represent three experiments. When shown, gray horizontal lines are limit of detection for assay. (F) IL-10 and IFN-γ production in media conditioned, without stimulation, for 24 h with total splenic leukocytes from d 0 or 3.5, or FACS-purified NK (CD49b+TCR-β−) and T (CD49b-TCR-β+) cells from d 3.5, high-dose-infected mice. Results represent two experiments. Values below detection are indicated by ϕ.

NK cells responding in spleens on d 3.5 after high-dose MCMV challenge were characterized for their expression of known NK cell markers (29). They had diminished expression of NKp46 and NK1.1. They expressed the CD49b NK cell marker but not the T cell receptor for antigen (i.e. TCR-β) (Supplemental Fig. 1A–D). The splenic proportions of CD49b+TCR-β− NK cells and Ly49H+ subsets in these populations were increasing during infection (Supplemental Fig. 1D, 1E). The NK (CD49b+TCR-β−) and T (CD49b-TCR-β+) cells were FACS purified from high-dose infected mice on 3.5 d, and their cytokine production in culture was compared to that of uninfected (d 0) or d 3.5 total leukocytes. Detectable IL-10 was only, and residual IFN-γ was predominantly, produced by the NK cells (Fig. 1F). To overcome difficulty in detecting IL-10 by flow cytometry, B6 mice reporting IL-10 with expression of green fluorescent protein (IL-10-GFP) (20) were analyzed. In comparison to T cell subsets, NK cells had high IL-10-GFP by d 3.5 of infection (Supplemental Fig. 1F). Analysis of all splenic leukocytes demonstrated that under these conditions of infection, the cells expressing IL-10-GFP were localized within subsets expressing NK cells markers (Supplemental Fig. 1G). Together, these results show that Wt mice with elevated and extended viral replication have systemic IL-10, and that NK cells are the major IL-10 producers.

IL-12 and IL-21 induction of IL-10 production by infection-conditioned NK cells

Leukocytes from mice, d 0 or d 3.5 high-dose-infected, were incubated in control media or with cytokines reported to induce IL-10, including IL-12, IL-21, IL-22, IL-23, and IL-27 (9, 13, 14, 16, 17, 30), for 24 h ex vivo. Two of the tested cytokines, IL-12 and IL-21, induced elevated IL-10 production by the d 3.5 populations (Fig. 2A). Only IL-12 significantly induced IFN-γ. When cells from IL-10-reporter mice were analyzed as either percentage positive and/or intensity of expression per cell, the IL-12 and IL-21 effects for IL-10-GFP expression were dramatic in NK (CD49b+TCR-β−) as compared T (CD49b-TCR-β+) cells and even higher in the Ly49H+TCR-β− subset (Fig. 2B). The consequences of cytokine exposure for IL-10 production using purified subsets in culture were consistent: only the d 3.5 NK cells responded to IL-12 or IL-21 with increased IL-10, and a distinct IFN-γ enhancing effect was mediated by IL-12 (Fig. 2C). High-dose infections of reporter mice with selective defects in responsiveness to IL-12 (Fig. 2D) or IL-21 (Fig. 2E) demonstrated IL-10 induction and NK cell IL-10-GFP expression in all infected mice having single defects. Therefore, NK cells conditioned during MCMV infection have an induced responsiveness to IL-12 and IL-21 for IL-10 expression, but these cytokines are not individually required for infection-induced NK cell IL-10 in vivo.

FIGURE 2. Cytokine induction of IL-10 ex vivo and in vivo.

FIGURE 2

Open in a new tab

B6 IL-10-GFP mice were uninfected or MCMV infected with 70,000 PFU for 3.5 d. NK cells were defined as CD49b+TCR-β− or Ly49H+TCR-β− and T cells as CD49b-TCR-β+. (A) IL-10 and IFN-γ production by total splenic leukocytes and (B) IL-10-GFP expression in NK and T cells, after 24 h stimulation in culture with IL-12, IL-21, IL-22, IL-23, or IL-27. Data in A and B are representative of three experiments. (C) IL-10 and IFN-γ production by total leukocytes or FACS-purified subsets from d 3.5 infected B6 mice was determined after 24 h stimulation with indicated cytokines. Data represent two experiments. Values below detection are indicated by ϕ. (D) Requirement for endogenous IL-12 in the 3.5 d NK cell IL-10 response were evaluated in B6 Wt mice rendered IL-12 deficient as a result of genetic mutation in gene for the p35 subunit (IL-12p35−/−) and in IL-10-GFP reporter mice rendered IL-12 deficient as a result of treatment with α-IL-12p40 in control mice. (E) Requirement for endogenous IL-21 in the NK cell IL-10 response were evaluated in mice rendered IL-21 deficient as a result of genetic mutation of the cytokine gene (IL-21−/−) or of the gene for the cytokine receptor (IL-21R−/−) in comparison to hemizygous littermates bred to also carry a IL-10-GFP reporter gene. The numbers in histograms indicate proportions of IL-10-GFP-expressing cells. Gray vertical lines indicate peak of basal GFP expression. Symbols in summary data are results with samples from individual mice. Mean ± s.d., of totals of 3–6 mice, are shown with bars. Studies are representative of, or pooled from, two to three independent experiments. *P<0.05, **P<0.01

Proliferation accompanies acquisition of the NK cell IL-10 response

The correlation of the IL-10 response with proliferation was examined independently of viral infection in culture studies using IL-2-driven expansion. Purified naïve NK cells, prepared from uninfected IL-10-GFP mice by negative selection, were examined. Because NK cells from uninfected mice require high IL-2 concentrations to be driven into expansion (31–33), IL-2 was added at 25 U/ml for control and at 1000 U/ml to induce proliferation. Fifty ng/ml of IL-12 was added at the initiation of culture (24 hr harvest), at 24 hr (48 hr harvest) or at 48 hr (72 hr harvest). Brefeldin A was added at 4 hr prior to harvest. The cells were prepared and examined for intracellular expression of IL-10-GFP and IFN-γ. With low-level IL-2, there was no discernable induction of IL-10-GFP but good induction of IFN-γ expression at the 24, 48 or 72 hr harvests. In the presence of high IL-2 concentrations, induction of IFN-γ was higher and the IL-10-GFP expression increased with time in culture (Fig. 3A). Purified naïve NK cells, again prepared from uninfected IL-10-GFP mice by negative selection, were labeled with the proliferation dye eFluor670 and cultured to examine the induction of IL-10-GFP and IFN-γ under the 72 hr harvest conditions. With the high IL-2 concentrations, the NK cell yields were approximately 4-fold higher, and the cells had undergone multiple divisions, with >7 subsets of eFluor670 dilution identifiable (Fig. 3B). In comparison to cells in low IL-2, higher IL-10-GFP expression was discernable with levels increasing upon further divisions to an apparent plateau at division 4 or 5. In contrast to IL-10, IFN-γ was dramatically induced following IL-12 exposure even in cells maintained under low IL-2 concentrations. The proportion and intensities of IL-10-GFP expressing cells were higher in the first division group from the high dose IL-2 condition, stayed high through 4 divisions, and then declined. The changing responsiveness resulted in NK cell populations with fewer divisions largely expressing IFN-γ, those with the most divisions largely expressing IL-10. The modification was independent of the Ly49H activating receptor because the shift to the IL-10-GFP response was observed in both Ly49H+ and Ly49H− subsets (Fig. 3C).

FIGURE 3. Cell division and changing NK cell responses ex vivo.

FIGURE 3

Open in a new tab

Purified NK cells from uninfected IL-10-GFP mice, prepared by negative selection and labeled with eFluor670 proliferation dye, were cultured in the presence of 25 or 1000 U/ml IL-2 for 24, 48 or 72 h, with 50 ng/ml IL-12p70 added at 24 h prior to harvest. Cells were collected and analyzed for intracellular expression of IL-10-GFP or IFN-γ with or without eFluor670 dilution. Representative flow cytometry plots are shown. (A) Expression of IL-10-GFP or IFN-γ after culture in IL-2 for 24, 48 or 72 h with IL-12 added at 24 h prior to harvest. (B) Cells were collected from samples at 72 h after culture with IL-2 with IL-12 added for the last 24 h and analyzed for eFluor670 dilution, along with intracellular expression of IL-10-GFP and IFN-γ. Representative flow cytometry plots of divisions and cytokine expression are shown. Summary data are replicate samples from a single experiment using pooled cells (mean ± s.d.) and are representative of three independent experiments. Statistical analyses evaluated the differences in proportions of cells expressing each cytokine after one as compared to 7 divisions. *P<0.05 (C) As in B but IL-10-GFP expression was characterized in the NK cell subsets either positive or negative for the Ly49H activating receptor. Representative flow cytometry plots of divisions and IL-10-GFP expression are shown. Summary data are replicate samples from a single experiment using pooled cells (mean ± s.d.), and are representative of two independent experiments.

To evaluate the association of proliferation with cytokine responses in vivo, mature naïve NK cells, purified from uninfected CD45.2 IL-10-GFP-reporter mice by negative selection, were labeled with the eFluor670 proliferation dye, and transferred to uninfected Ly49H-deficient B6 CD45.1. The recipient mice were then MCMV infected. Examination of the levels of eFluor670 expression demonstrated that although the transferred cells readily expressed IFN-γ at d 1.5 of infection, most of the cells had not yet undergone proliferation and expressed only low levels of IL-10-GFP (Fig. 4A). Proliferation was enhanced by d 2.5 and dramatically extended by d 3.5 with many of the transferred NK cells having undergone extensive numbers of divisions. Although they expressed less IFN-γ, the NK cells expressed higher levels of IL-10-GFP at d 2.5 and d 3.5 of infection (results summarized in Fig. 4B). Together, these results show that the NK cell IL-10 response is associated with extensive proliferation either under in vitro conditions independent of infection or in vivo during infection.

FIGURE 4. Cell division and cytokine expression during MCMV infection in vivo.

FIGURE 4

Open in a new tab

Purified NK cells, prepared by negative selection, from uninfected IL-10-GFP (CD45.2) mice were labeled with eFluor670 and i.v. transferred to Ly49H−/− (CD45.1) mice. Recipient mice were then uninfected or low-dose infected and harvested at d 0, 1.5, 2.5 or 3.5. Splenic leukocytes were analyzed for intracellular IL-10-GFP expression and eFluor670 dilution among donor CD45.2 cells. (A) Representative flow cytometric plots are shown. Numbers in histograms are proportion of cells in gate. When shown, gray vertical lines show peak of basal expression. (B) Summary data are from three independent experiments totaling 4–10 mice per time point with each point representing a single mouse (mean ± s.d.). Comparison of d 0 to d 2.5 or d 3.5 gave differences significant to ***P<0.0001 for all parameters. Differences between d 1.5 and d2.5 or d 3.5 are shown, **P<0.001, ***P<0.0001. Differences in each parameter (proliferation, IL-10, and IFN-γ) are significant by one-way ANOVA across all groups with ***P<0.0001.

Intrinsic changes in NK cells to allow IL-10 response

To define intrinsic as compared to extrinsic influences, adoptive transfer experiments were carried out with cells isolated from d 0, d 1-infected or d 3-infected IL-10-GFP-reporter CD45.2 B6 mice and then delivered to Wt CD45.1 B6 mice that were d 0 or MCMV infected at d 1 or 3 prior to transfer (Fig. 5A). The donor mice were infected with 70,000 PFU, and recipient mice were challenged with 5,000 PFU. Transferred cells were allowed to experience endogenous stimuli in recipient mice for one day, harvested and analyzed for IL-10-GFP expression in gated NK cells among CD45.2 donor populations. When transferred to d 0 environments, NK cells from d 3-infected (noted by red lines in histograms) showed modest residual IL-10-GFP expression. Dramatic IL-10-GFP induction was observed when NK cells from d 3-infected donors were transferred into d 1 recipients, whereas NK cells from d 0 or d 1 donors showed minimal expression. The IL-10-GFP levels were intensified in Ly49H+TCR-β− as compared to CD49b+TCR-β− cells (Fig. 5A). In d 3 recipients, expression was detectable but reduced as compared to d 1 recipients. Parallel experiments were carried out examining IFN-γ. The NK cell populations from d 0, d1, or d 3 infected mice all had IFN-γ induction when transferred into d 1-infected mice (Fig. 5B). To test the role for endogenous IL-12 in supporting the responses, the effects of α-IL-12p40 treatment were evaluated. Blocking IL-12 abolished induction of IL-10-GFP and IFN-γ in NK cells prepared from d 3 and transferred into d 1 MCMV-infected mice (Fig. 5C). Hence, NK cells are intrinsically pre-equipped for IFN-γ induction in response to IL-12 but acquire the ability to respond with IL-10 as infection progresses. When they experience IL-12 in an in vivo environment, they now produce both cytokines.

FIGURE 5. Intrinsic and extrinsic influences on NK cell responsiveness for IL-10 and IFN-γ expression.

FIGURE 5

Open in a new tab

Donor splenic leukocytes from B6 IL-10-GFP (CD45.2) mice, d 0 or infected with high dose MCMV for 1 or 3 d, were transferred into B6 (CD45.1) recipients that were either d 0, or infected with lose dose virus for 1 or 3 d. Recipient mice were left for one day before harvesting and analyzing IL-10-GFP (A) or IFN-γ (B) expression within gated donor (CD45.2) NK cells. Filled gray histograms indicate basal cytokine expression in transferred d 0 cells into d 0 mice. Blue, green, and red histograms indicate expression in donor NK cells prepared from d 0, d 1-infected, and d 3-infected IL-10-GFP mice, respectively. Summary data are from three experiments, with 4–6 mice per group total. (C) As in A and B with the modification that donor leukocytes from d 3-infected IL-10-GFP mice were adoptively transferred into d 1 B6 recipients that had received 750 ug α-IL-12p40 or control Ab 4 h before infection. Results are from two experiments, 5 mice per group total. (A, B) Numbers on histograms are proportion of cells within gate. Representative data are shown. Results are summarized with symbols representing individual mice. Statistical significances are shown, **P<0.01, ***P<0.0001. For the Figs. 5A summary data, the results are also significant by one-way ANOVA with ***P<0.0001.

Chromosomal changes in the NK cell IL-10 gene during infection

One mechanism with the potential for regulating access to the IL-10 gene is epigenetic modification of associated histones to control access of transcriptional machinery (25, 34). In particular, histone 3 modifications assessed as K4 trimethylation (m3) or K36 trimethylation (m3) are indicative of euchromatin with an “open” structure, whereas K27 trimethylation (m3) is indicative of a “closed” structure. To evaluate the possible involvement of epigenetic modification in the control of NK cell IL-10 expression during MCMV infection, chromatin immunoprecipitation was performed. Splenic leukocytes were harvested from d 0, 1.5, or 3.5 MCMV-infected mice, and subjected to differential centrifugation and magnetic bead negative selection to enrich for NK cells, yielding high purity and sufficient numbers for study. Cell pellets were cross-linked and antibodies specific for the histone 3 activation marks K4m3 and K36m3, and the inhibitory mark K27m3 were used. The two activating methylations, K4m3 and K36m3, were largely absent from the IL-10 gene at d 0 in NK cells, and increased dramatically as infection progressed. The K27m3 inhibitory methylation was present in d 0 and 1.5 NK cells, but absent in d 3.5 NK cells (Fig. 6A). In contrast, the IFN-γ gene has been reported to be opened in NK cells from uninfected mice (35), and it had an the opened profile in the NK cells purified from d 0, as well as d 1.5 and d 3.5 MCMV-infected mice (Fig. 6B). The gene for the myogenic differentiation 1 (Myod1) has been reported to be repressed by K27me3 in cells other than muscle cells, including ES, and CD4 T cells (36, 37), and all three NK cell populations in this study had a closed Myod1 profile (Fig. 6C) genes were at all times examined. These data demonstrate epigenetic changes in the NK cell IL-10 gene suggesting that unlike the IFN-γ and Myod1 genes, it is being modified during the conditions of infection to become more accessible for expression at times only after significant proliferation had occurred in vivo.

FIGURE 6. Increased accessibility of the IL-10 locus during the course of MCMV infection.

FIGURE 6

Open in a new tab

Distribution of histone marks were assessed by chromatin immunoprecipitation and massive parallel sequencing of purified NK cells ex vivo from Wt B6 uninfected (day 0) or MCMV infected for 1.5 and 3.5 days. NK cells prepared from leukocytes isolated from 20 (Day 0), 40 (Day 1.5) and 40 (Day 3.5) mice were purified to ~80%. Results using day 0 and day 3.5-infected mice were repeated twice. Histone modifications assessed are H3K4 trymethylation (red), H3K27 trymentylation (blue) and H3K36 trimethylation (green). Results are shown for the IL-10 (A), IFN-γ (B), and Moyd1 (C) genes.

Proliferation requirement for the changing NK cell response

To establish the requirement for proliferation, leukocytes from d 0 or d 1.5, 2.5 and 3.5 high-dose-MCMV-infected IL-10-GFP-reporter mice were harvested, labeled with eFluor670, control-treated or treated with Mitomycin C (MMC) to block their ability to proliferate, and then cultured with high dose IL-2 for 48 hr with or without addition of IL-12 for the last 24 hr. The NK cells, identified as CD49b+TCR-β−, prepared from d 0 and d 1.5 MCMV-infected mice were induced to proliferate and respond to IL-12 with IL-10-GFP expression, and this was dramatically inhibited when the cells were treated with MMC (Fig. 7A). In contrast, cells harvested on d 2.5 or 3.5 after infection, the times associated with extensive proliferation of the populations in vivo, were capable of responding to IL-12 with IL-10-GFP expression whether or not proliferation had been blocked by MMC. The MMC effects for inhibiting IL-10-GFP expression in response to IL-12 were highly significant using the populations from d 0 or d 1.5-infected mice but insignificant using the populations prepared on d 2.5 or 3.5 of infection (results summarized in Fig. 7B). Thus, NK cells acquire an IL-10 response to IL-12 after division, and the shift requires the ability to proliferate but is proliferation independent if the cells have already expanded during infection.

FIGURE 7. Proliferation requirement for changing NK cell response.

FIGURE 7

Open in a new tab

Splenic leukocytes were isolated from IL-10-GFP mice that were uninfected (day 0), or infected with high-dose MCMV for 1.5, 2.5 or 3.5 d. Cells were labeled with eFluor670, with or without treatment with 50 µg/ml MMC to block proliferation, and cultured in the presence of 1000 U/ml IL-2 for 48 h, with or without addition of 50ng/ml IL-12p70 24 h prior to harvest. NK cells were identified as CD49b+TCR-β− and analyzed for eFluor670 dilution and intracellular IL-10-GFP expression. Representative histograms are shown (A). Summary data, collected from three experiments with totals of 12, 6, 2, and 8 respectively at d 0, 1.5, 2.5 and 3.5, are shown with symbols representing results from individual mice (mean ± S.D.) ***P<0.0001 (B). For the summary data, the results are also significant by one-way ANOVA with ***P<0.0001.

Discussion

This report conclusively demonstrates proliferation-dependent conditioning of NK cells (Fig. 7). During sustained viral infection, NK cells acquire the ability to produce IL-10, and shift the response induced by IL-12 from IFN-γ only to IFN-γ and IL-10. The shift to an ability to produce IL-10 is important in contributing to the regulation of CD8 T cell responses to infection (4). It is accompanied by epigenetic changes in the IL-10 gene supporting its expression (Fig. 6). Thus, one answer to the question of why NK cell proliferate when their basal frequencies are high is provided, and a previously unappreciated role for proliferation in shaping innate cell “preparedness” for particular functions is reported.

Linking proliferation to switching NK cells from inflammatory to regulatory provides a pathway for taking advantage of these high frequency innate cells to apply negative pressure on adaptive immunity. The results suggest that the previously reported NK cell IL-10 responses observed in MCMV-infected mice (4, 10, 12) and in HCV-infected humans (8) as well as during mouse infections with the parasitic agent Toxoplasma gondii (11, 38) are delivered by cells that have proliferated. The intrinsic changes reported here result in the ability of NK cells to express IL-10 in response to different stimuli, i.e. IL-21 as well as IL-12 (Figs. 4 and 7). Endogenous IL-12 does contribute to the NK cell IL-10 response to the parasitic agent (11) but is not required for the NK cell IL-10 elicited during high-dose MCMV infection (Fig. 2D). IL-21 is also not required (Fig. 2E), and the IL-2-supported proliferation-dependent induction of NK cell responsiveness to IL-12 for IL-10 expression shows that stimulation through particular NK activating receptors is not a prerequisite (Fig. 3). Thus, once NK cells are prepared to express IL-10, they may do so in response to a variety of stimuli if any are present in the environment.

In addition to demonstrating a novel role for proliferation, the studies help explain a complicated literature on IL-12 induction of IL-10 (30, 39). The earlier work noted an IL-10 NK cell response to IL-12 in longer-term cultures and/or under conditions of IL-2 exposure that support NK cell proliferation. The demonstrations here that IL-2 concentrations insufficient to support NK cell proliferation (Fig. 3), and IL-2 concentrations sufficient to support proliferation but NK cells blocked in their ability to proliferate (Fig. 7), fail to develop an IL-10 response to IL-12 unless they had previously divided in vivo. Thus, these studies establish the role of proliferation in the changing response. In addition, they provide insights into the biological role for the changing response, i.e. to use NK cells for regulatory functions under conditions of their sustained stimulation for proliferation.

The results suggest consequences of proliferation that have been overlooked in the shaping of other cell functions. Similar to NK cells, innate monocyte/macrophage populations are at high frequencies, and macrophage proliferation has been observed in situ (40). Thus, macrophage proliferation might also promote conditioning of these cells to deliver differential effects. Dendritic cells have been reported to have epigenetic changes consistent with limiting IL-12 expression during their repopulation following sepsis (41), but the role for proliferation in the development of the cells has not been examined.

The high frequencies of NK cells constitutively found in uninfected mice are in contrast to the low frequencies of antigen-specific T cells under naïve conditions. Stimulation through the TCR is required for CD4 or CD8 T cell expansion to numbers sufficient for defense. In the context of infections resulting in extensive proliferation, CD8 T cell expression of complex immune enhancing factors is narrowed and an IL-10 response can be acquired (42–45). These shifts do parallel the NK cell changes, but the suggested roles for proliferation in regulating CD4 or CD8 T cell cytokine responses have been based on correlations of cytokine expression with dilution of a proliferation dye after stimulation through the TCR (45, 46). Because the shift to NK cell IL-10 expression is shown here using IL-2 to support proliferation ex vivo (Fig. 3) as well as MCMV infection to support proliferation in vivo (Fig. 4), the results document changing function in the absence of viral ligands for activating receptors. Moreover, the experiments using Mitomycin C to block division (Fig. 7) establish that the change is dependent on the proliferation event.

Cell division and differentiation are a complex processes that dependent on metabolic changes and accumulation of materials to complete the process (47–49). The experiments carried out to date are only beginning to define the specific steps in the processes uniquely linked to changing function (50). In addition to demonstrating the importance for proliferation in the evolving function, however, our results document epigenetic changes in the NK cells responding to infection such that the IL-10 gene acquires histone modifications indicative of an open state for transcription and expression (Fig. 6). These data indicate that the intrinsic NK cell changes supported by the conditions of proliferation include those required for epigenetic modification of the IL-10 gene. The magnitude of the changes captured here is somewhat surprising and reveals previously unsuspected dynamics in genetic modification. Given the demonstration of NK cell responses through periods overlapping adaptive immunity and under “memory” conditions (51–53) and the question of inheritance of epigenic modifications (54), important questions about the short- and long-term consequences of the effects are suggested by the data. The fact that the shift to IL-12 responsiveness for IL-10 expression is induced in culture conditions without purposeful addition of other proinflammatory cytokines indicates that these are not required for the conditioning of intrinsic NK cell responses to induce IL-10 expression. Their roles in promoting histone modification for stable responses, however, remain to be tested. Likewise, the dissection of possible different paths to proliferation, including stimulation through the activating receptors and/or with growth factors such as IL-2, for stable genetic modification remain to be elucidated. Thus, work beyond the scope of this report is suggested by the results.

When IL-2 was used to support proliferation in culture (Fig. 3), the proportions of Ly49H cells remained constant, and both Ly49H+ and Ly49H− populations acquired the ability to express IL-10. During the MCMV infection, NK cell proliferation is induced through cytokine-dependent and Ly49H-independent pathways at early times after infection, but the proportions of Ly49H+ cells increase at later times because of stimulation resulting from expression of a viral ligand (2). In the experiments reported here, the NK cell subsets went from 40% to 80% Ly49H+ cells during infection (Supplemental Fig. 1). The subset responded more dramatically to IL-12 stimulation for IL-10 expression (Fig. 2), but both Ly49H+ and Ly49H− populations expressing IL-10 could be identified during infection (Supplemental Fig. 1). It is not clear whether or not stimulation through the Ly49H receptor provided additional signals for responsiveness or simply additional support for extended proliferation. Taken together, however, the results demonstrate that an Ly49H signal is not required to drive NK cells to IL-10 expression.

In conclusion, these studies define a novel role for proliferation in shaping innate cell “preparedness” for responses, and demonstrate intrinsic molecular genetic modifications accompanying the alterations in function supported by cell division. They provide novel insights into the dynamic events controlling NK cell functions as needed and suggest a more general role for proliferation in regulating functions of multiple immune cell subsets.

Supplementary Material

1

Acknowledgements

The authors thank Dr. Guiseppe Sciume for advice on preparing cell populations, Dr. Mercedes Rincon for gift of mice, and Kwang-Sin Kim, D. Ashley Feldman, and Delia A. Demers for technical assistance.

Footnotes

3

The work was supported by the National Institutes of Health, USA and by a Graduate Assistance in Area of National Need (GAANN) Award from the Department of Education, USA.

4

Abbreviations used in this article: Murine cytomegalovirus, MCMV; myogenic differentiation 1, Myod1

Disclosures

The authors have no financial conflicts of interests.

References

  • 1.Biron CA, Sonnenfeld G, Welsh RM. Interferon induces natural killer cell blastogenesis in vivo. J. Leuk. Biol. 1984;35:31–37. doi: 10.1002/jlb.35.1.31. [DOI] [PubMed] [Google Scholar]
  • 2.Dokun AO, Kim S, Smith HR, Kang HS, Chu DT, Yokoyama WM. Specific and nonspecific NK cell activation during virus infection. Nat. Immunol. 2001;2:951–956. doi: 10.1038/ni714. [DOI] [PubMed] [Google Scholar]
  • 3.Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei XQ, Liew FY, Caligiuri MA, Durbin JE, Biron CA. Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. J. Immunol. 2002;169:4279–4287. doi: 10.4049/jimmunol.169.8.4279. [DOI] [PubMed] [Google Scholar]
  • 4.Lee SH, Kim KS, Fodil-Cornu N, Vidal SM, Biron CA. Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection. J. Exp. Med. 2009;206:2235–2251. doi: 10.1084/jem.20082387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Vidal SM, Khakoo SI, Biron CA. Natural killer cell responses during viral infections: flexibility and conditioning of innate immunity by experience. Curr. Opin. Virol. 2011;1:497–512. doi: 10.1016/j.coviro.2011.10.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Orange JS, Biron CA. An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections. J. Immunol. 1996;156:1138–1142. [PubMed] [Google Scholar]
  • 7.Peritt D, Robertson S, Gri G, Showe L, Aste-Amezaga M, Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets. J. Immunol. 1998;161:5821–5824. [PubMed] [Google Scholar]
  • 8.De Maria A, Fogli M, Mazza S, Basso M, Picciotto A, Costa P, Congia S, Mingari MC, Moretta L. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patients. Eur. J. Immunol. 2007;37:445–455. doi: 10.1002/eji.200635989. [DOI] [PubMed] [Google Scholar]
  • 9.Grant LR, Yao ZJ, Hedrich CM, Wang F, Moorthy A, Wilson K, Ranatunga D, Bream JH. Stat4-dependent, T-bet-independent regulation of IL-10 in NK cells. Genes Immun. 2008;9:316–327. doi: 10.1038/gene.2008.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Maroof A, Beattie L, Zubairi S, Svensson M, Stager S, Kaye PM. Posttranscriptional regulation of II10 gene expression allows natural killer cells to express immunoregulatory function. Immunity. 2008;29:295–305. doi: 10.1016/j.immuni.2008.06.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Perona-Wright G, Mohrs K, Szaba FM, Kummer LW, Madan R, Karp CL, Johnson LL, Smiley ST, Mohrs M. Systemic but not local infections elicit immunosuppressive IL-10 production by natural killer cells. Cell Host Microbe. 2009;6:503–512. doi: 10.1016/j.chom.2009.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gaddi PJ, Crane MJ, Kamanaka M, Flavell RA, Yap GS, Salazar-Mather TP. IL-10 mediated regulation of liver inflammation during acute murine cytomegalovirus infection. PLoS One. 2012;7:e42850. doi: 10.1371/journal.pone.0042850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Stumhofer JS, Silver JS, Laurence A, Porrett PM, Harris TH, Turka LA, Ernst M, Saris CJ, O'Shea JJ, Hunter CA. Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. Nat. Immunol. 2007;8:1363–1371. doi: 10.1038/ni1537. [DOI] [PubMed] [Google Scholar]
  • 14.Brady J, Hayakawa Y, Smyth MJ, Nutt SL. IL-21 induces the functional maturation of murine NK cells. J. Immunol. 2004;172:2048–2058. doi: 10.4049/jimmunol.172.4.2048. [DOI] [PubMed] [Google Scholar]
  • 15.Saraiva M, Christensen JR, Veldhoen M, Murphy TL, Murphy KM, O'Garra A. Interleukin-10 production by Th1 cells requires interleukin-12-induced STAT4 transcription factor and ERK MAP kinase activation by high antigen dose. Immunity. 2009;31:209–219. doi: 10.1016/j.immuni.2009.05.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, Cua DJ. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat. Immunol. 2007;8:1390–1397. doi: 10.1038/ni1539. [DOI] [PubMed] [Google Scholar]
  • 17.Saraiva M, O'Garra A. The regulation of IL-10 production by immune cells. Nat. Rev. Immunol. 2010;10:170–181. doi: 10.1038/nri2711. [DOI] [PubMed] [Google Scholar]
  • 18.Saraiva M, Christensen JR, Tsytsykova AV, Goldfeld AE, Ley SC, Kioussis D, O'Garra A. Identification of a macrophage-specific chromatin signature in the IL-10 locus. J. Immunol. 2005;175:1041–1046. doi: 10.4049/jimmunol.175.2.1041. [DOI] [PubMed] [Google Scholar]
  • 19.Shoemaker J, Saraiva M, O'Garra A. GATA-3 directly remodels the IL-10 locus independently of IL-4 in CD4+ T cells. J. Immunol. 2006;176:3470–3479. doi: 10.4049/jimmunol.176.6.3470. [DOI] [PubMed] [Google Scholar]
  • 20.Kamanaka M, Kim ST, Wan YY, Sutterwala FS, Lara-Tejero M, Galan JE, Harhaj E, Flavell RA. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity. 2006;25:941–952. doi: 10.1016/j.immuni.2006.09.013. [DOI] [PubMed] [Google Scholar]
  • 21.Mattner F, Magram J, Ferrante J, Launois P, Di Padova K, Behin R, Gately MK, Louis JA, Alber G. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 1996;26:1553–1559. doi: 10.1002/eji.1830260722. [DOI] [PubMed] [Google Scholar]
  • 22.Dienz O, Eaton SM, Bond JP, Neveu W, Moquin D, Noubade R, Briso EM, Charland C, Leonard WJ, Ciliberto G, Teuscher C, Haynes L, Rincon M. The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4+ T cells. J. Exp. Med. 2009;206:69–78. doi: 10.1084/jem.20081571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Fodil-Cornu N, Lee SH, Belanger S, Makrigiannis AP, Biron CA, Buller RM, Vidal SM. Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex. J. Immunol. 2008;181:6394–6405. doi: 10.4049/jimmunol.181.9.6394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Brandt K, Bulfone-Paus S, Foster DC, Ruckert R. Interleukin-21 inhibits dendritic cell activation and maturation. Blood. 2003;102:4090–4098. doi: 10.1182/blood-2003-03-0669. [DOI] [PubMed] [Google Scholar]
  • 25.Zang C, Schones DE, Zeng C, Cui K, Zhao K, Peng W. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics. 2009;25:1952–1958. doi: 10.1093/bioinformatics/btp340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Orange JS, Biron CA. Characterization of early IL-12, IFN-alphabeta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J. Immunol. 1996;156:4746–4756. [PubMed] [Google Scholar]
  • 27.Ruzek MC, Miller AH, Opal SM, Pearce BD, Biron CA. Characterization of early cytokine responses and an interleukin (IL)-6-dependent pathway of endogenous glucocorticoid induction during murine cytomegalovirus infection. J. Exp. Med. 1997;185:1185–1192. doi: 10.1084/jem.185.7.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Delale T, Paquin A, Asselin-Paturel C, Dalod M, Brizard G, Bates EE, Kastner P, Chan S, Akira S, Vicari A, Biron CA, Trinchieri G, Briere F. MyD88-dependent and -independent murine cytomegalovirus sensing for IFN-alpha release and initiation of immune responses in vivo. J. Immunol. 2005;175:6723–6732. doi: 10.4049/jimmunol.175.10.6723. [DOI] [PubMed] [Google Scholar]
  • 29.Huntington ND, Vosshenrich CA, Di Santo JP. Developmental pathways that generate natural-killer-cell diversity in mice and humans. Nat. Rev. Immuno.l. 2007;7:703–714. doi: 10.1038/nri2154. [DOI] [PubMed] [Google Scholar]
  • 30.Peritt D, Aste-Amezaga M, Gerosa F, Paganin C, Trinchieri G. Interleukin-10 induction by IL-12: a possible modulatory mechanism? Annals New York Acad. Sciences. 1996;795:387–389. doi: 10.1111/j.1749-6632.1996.tb52701.x. [DOI] [PubMed] [Google Scholar]
  • 31.Biron CA, Young HA, Kasaian MT. Interleukin 2-induced proliferation of murine natural killer cells in vivo. J. Exp. Med. 1990;171:173–188. doi: 10.1084/jem.171.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kasaian MT, Biron CA. Cyclosporin A inhibition of interleukin 2 gene expression, but not natural killer cell proliferation, after interferon induction in vivo. J. Exp. Med. 1990;171:745–762. doi: 10.1084/jem.171.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Lee SH, Fragoso MF, Biron CA. Cutting edge: a novel mechanism bridging innate and adaptive immunity: IL-12 induction of CD25 to form high-affinity IL-2 receptors on NK cells. J. Immunol. 2012;189:2712–2716. doi: 10.4049/jimmunol.1201528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Zhou VW, Goren A, Bernstein BE. Charting histone modifications and the functional organization of mammalian genomes. Nature reviews. Genetics. 2011;12:7–18. doi: 10.1038/nrg2905. [DOI] [PubMed] [Google Scholar]
  • 35.Stetson DB, Mohrs V, Reinhardt RL, Baron JL, Wang ZE, Gapin L, Kronenberg M, Locksley RM. Constitutiive cytokine mRNA mark natural killer (NK) and NK T cells poised for rapid effector function. J. Exp. Med. 2003;198:1069–1075. doi: 10.1084/jem.20030630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Lee IL, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K-I, Koseki H, Fuchikami T, Abe K, Murray HL, Zucker JP, Yuan B, Bell GW, Herbolsheimer E, Hannett NM, Sun K, Odom DT, Otte AP, Bartel DP, Melton DA, Gifford DK, Jaenisch R, Young RA. Control of developmental regulators by polycomb in human embryonic cells. Cell. 2006;125:301–313. doi: 10.1016/j.cell.2006.02.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Wei G, Wei L, Zhu J, Zang C, Hu-Li J, Yao Z, Cui K, Kanno Y, Roh T-Y, Watford WT, Schones DE, Peng W, Sun H-w, Paul WE, O'Shea JJ, Zhao K. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiatin CD4+ T cells. Immunity. 2009;30:155–167. doi: 10.1016/j.immuni.2008.12.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wagage S, John B, Krock BL, Hall AO, Randall LM, Karp CL, Simon MC, Hunter CA. The aryl hydrocarbon receptor promotes IL-10 production by NK cells. J. Immuol. 2014;192:1661–1670. doi: 10.4049/jimmunol.1300497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Mehrotra PT, Donnelly RP, Wong S, Kanegane H, Gerem ew A, Mostowski HS, Furuke K, Siegel JP, Bloom ET. Production of IL-10 by human natural killer cells stimulated with IL-2 and/or IL-12. J. Immunol. 1998;160:2637–2644. [PubMed] [Google Scholar]
  • 40.Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, van Rooijen N, MacDonald AS, Allen JE. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science. 2011;332:1284–1288. doi: 10.1126/science.1204351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wen H, Dou Y, Hogaboam CM, Kunkel SL. Epigenetic regulation of dendritic cell-derived interleukin-12 facilitates immunosuppression after a severe innate immune response. Blood. 2008;111:1797–1804. doi: 10.1182/blood-2007-08-106443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Jin HT, Anderson AC, Tan WG, West EE, Ha SJ, Araki K, Freeman GJ, Kuchroo VK, Ahmed R. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc. Natl. Acad. Sciences, USA. 2010;107:14733–14738. doi: 10.1073/pnas.1009731107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Sun J, Madan R, Karp CL, Braciale TJ. Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10. Nat. Medicine. 2009;15:277–284. doi: 10.1038/nm.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine design. Nat. Rev. Immunol. 2008;8:247–258. doi: 10.1038/nri2274. [DOI] [PubMed] [Google Scholar]
  • 45.Denton AE, Russ BE, Doherty PC, Rao S, Turner SJ. Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+ T cells. Proc. Natl. Acad. Sciences, USA a. 2011;108:15306–15311. doi: 10.1073/pnas.1112520108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Gett AV, Hodgkin PD. Cell division regulated the T cell cytokine repertoire, revealing a mechanism underlying class regulation. Proc. Natl. Acad. Sci. USA. 1998;95:9488–9493. doi: 10.1073/pnas.95.16.9488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, Oda M. Eukaryotic chromosome DNA replication: where, when, and how? Annu. Rev. Biochem. 2010;79:89–130. doi: 10.1146/annurev.biochem.052308.103205. [DOI] [PubMed] [Google Scholar]
  • 48.Powell JD, Pollizzi KN, Heikamp EB, Horton MR. Regulation of immune responses by mTOR. Annu. Rev Immunol. 2012;30:39–68. doi: 10.1146/annurev-immunol-020711-075024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA. PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells. J. Exp. Med. 2012;209:2441–2453. doi: 10.1084/jem.20112607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Avni O, Rao A. T cell differentiation: a mechanistic view. Curr. Opin. Immunol. 2000;12:6654–657. doi: 10.1016/s0952-7915(00)00158-8. [DOI] [PubMed] [Google Scholar]
  • 51.O'Leary JG, Goodarzi M, Drayton DL, von Andrian UH. T cell- and B cell-independent adaptive immunity mediated by natural killer cells. Nat. Immunol. l. 2006;7:507–516. doi: 10.1038/ni1332. [DOI] [PubMed] [Google Scholar]
  • 52.Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature. 2009;457:557–561. doi: 10.1038/nature07665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Paust S, Gill HS, Wang BZ, Flynn MP, Moseman EA, Senman B, Szczepanik M, Telenti A, Askenase PW, Compans RW, von Andrian UH. Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses. Nat. Immuno.l. 2010;11:1127–1135. doi: 10.1038/ni.1953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Carone BR, Fauquier L, Habib N, Shea JM, Hart CE, Li R, Bock C, Li C, Gu H, Zamore PD, Meissner A, Weng Z, Hofmann HA, Friedman N, Rando OJ. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell. 2010;143:1084–1096. doi: 10.1016/j.cell.2010.12.008. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS592271-supplement-1.pdf (1MB, pdf)

RESOURCES