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DOI: 10.1002/eji.201445209

Activated NKT cells imprint NK-cell differentiation, functionality and education Peggy Riese ∗1 , Stephanie Trittel ∗1,2 , Tobias May3 , Luka Cicin-Sain2 , Benedict J. Chambers4 and Carlos A. Guzm´ an1 1

Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Junior Research Group Immune Aging and Chronic Infections, Helmholtz Centre for Infection Research, Braunschweig, Germany 3 Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany 4 Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden NK cells represent a vital component of the innate immune system. The recent discoveries demonstrating that the functionality of NK cells depends on their differentiation and education status underscore their potential as targets for immune intervention. However, to exploit their full potential, a detailed understanding of the cellular interactions involved in these processes is required. In this regard, the cross-talk between NKT cells and NK cells needs to be better understood. Our results provide strong evidence for NKT cell-induced effects on key biological features of NK cells. NKT-cell activation results in the generation of highly active CD27high NK cells with improved functionality. In this context, degranulation activity and IFNγ production were mainly detected in the educated subset. In a mCMV infection model, we also demonstrated that NKT-cell stimulation induced the generation of highly functional educated and uneducated NK cells, crucial players in viral control. Thus, our findings reveal new fundamental aspects of the NKT-NK cell axis that provide important hints for the manipulation of NK cells in clinical settings.

Keywords: αGalCerMPEG r mCMV r NK-cell differentiation r NK-cell education



r

NKT cells

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction NK cells represent a first line of defence against transformed or viral-infected cells by exerting immune regulatory and cytotoxic functions [1]. It was long believed that NK cells are a homogenous and short-lived innate lymphocyte population that retains their fixed phenotypic and functional characteristics. However, findings of a continuous NK-cell differentiation process at steady-

Correspondence: Dr. Peggy Riese e-mail: [email protected]  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

state conditions underscore their, up to now underestimated, multifunctional properties [2–4]. Several groups demonstrated that human and mouse NK cells represent a heterogeneous population that can be divided into different subsets with distinct functional properties [5–8]. In this context, CD27 and Mac-1 have been identified as key markers to dissect murine NK cells. CD27high Mac-1high NK cells display enhanced responsiveness, whereas the CD27low Mac-1high subset represents NK cells with a higher activation

∗

These authors contributed equally to this work.

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threshold [9, 10]. The similarities between CD27 and Mac-1 expressing murine NK cells and human CD56bright and CD56dim NK-cell subsets enables a phenotypic and functional characterization comparable to the human system [11, 12]. To ensure self-tolerance, NK-cell functionality is controlled by a process termed “education.” Educated NK cells expressing inhibitory receptors binding to self-MHC molecules are more responsive to stimulation, whereas uneducated NK cells lacking self-MHC class I receptors are considered to be hypo-responsive [13–15]. The process of education appears to be tuneable, depending on the net signaling received by NK cells [16]. This introduces a dynamic aspect of NK-cell development, as well as a new level of functional heterogeneity of distinct NK-cell subsets. The functional heterogeneity of NK cells is further proved by the discovery of their adaptive immune features [17–19]. This renders them interesting targets for clinical application. However, the exploitation of the true potential of NK cells requires a more detailed understanding of their biology, as well as tools to modify their functional properties. CD1d-restricted NKT cells represent an innate immune cell population known to activate NK cells. Treatment of NKT cells with the CD1d-binding agonist α-galactosylceramide (αGalCer) leads to the secretion of IFNγ and proliferation of NK cells that in turn results in anti-tumor and anti-viral activity [20–24]. However, the impact of NKT cells on NK-cell differentiation and education has not yet been fully elucidated. Thus, in the present study the potential of activated NKT cells to modulate fundamental NK-cell properties was investigated using a pegylated derivative of αGalCer (αGalCerMPEG). This compound has been shown to exhibit superior physicochemical and immune modulatory properties as compared to the parental compound [25]. We addressed the influence of αGalCerMPEG-activated NKT cells on the differentiation status of NK cells, as well as their implications in the process of NK-cell education and the subsequent functional properties. The functional impact of activated NKT cells on NK-cell features was further investigated in a mCMV infection model to evaluate their potential role in clinical settings.

Results Stimulation of NKT cells results in an increased number of CD27high NK cells To determine whether stimulation of NKT cells with αGalCerMPEG affects splenic lymphocytes and NK cells, the absolute NK-cell numbers were analyzed at different time points after s.c. administration. Increased absolute lymphocyte numbers were detected 24 and 72 h after NKT-cell stimulation (Fig. 1A). An initial decline of splenic NK cells was observed that started to recover 72 h after αGalCerMPEG administration (Fig. 1B). This decline was not due to activation-induced internalization of NKp46 (Supporting Information Fig. 1). In parallel, 24 and 72 h after NKT-cell activation an increased level of liver NK cells was observed (data not shown). To investigate if specific NK-cell subsets, defined by the expression of  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Innate immunity

CD27 and Mac-1, are influenced by αGalCerMPEG-activated NKT cells, the distribution of NK-cell subsets was assessed. Increased absolute numbers of CD27high Mac-1high expressing NK-cell subsets were detected as compared to controls. The increment was accompanied by a decreased absolute number of CD27low Mac1high expressing NK cells (Fig. 1C). The analysis of the relative frequencies of the distinct NK-cell subsets was consistent with these observed changes. An increased frequency of CD27high Mac-1low (30%) and CD27high Mac-1high (40%) expressing NK cells were detected as compared to controls (12 and 20%), respectively (Fig. 1D, E). In vitro studies in which bone marrow-derived DCs pulsed with αGalCerMPEG were cocultured with spleen-derived NKT and NK cells further supported the concept that activated NKT cells trigger the expansion of the CD27high Mac-1high NKcell subset (Fig. 1F). To assess whether activation of NKT cells with αGalCerMPEG also results in enhanced proliferative capacity of specific lymphocyte subsets, an in vivo proliferation assay was performed. For this, bromodeoxyuridine (BrdU) incorporation was analyzed 72 h after αGalCerMPEG administration. A strong increase in the percentage of BrdU+ NK cells (40%) was detected in αGalCerMPEG-treated animals when compared to the control group (5%). In contrast, the proliferation of CD3+ cells was not significantly affected (Fig. 1G). Dissection of BrdU+ splenic NK cells into the distinct differentiation stages showed the highest proliferative capacity following treatment with αGalCerMPEG in the CD27high Mac-1low (35%) and CD27high Mac-1high (40%) subsets (Fig. 1H). Thus, the obtained results clearly indicate a NKT-cell-induced enhanced proliferative capacity of NK cells, especially of the CD27high subsets. Various aspects of NK-cell development, homeostasis, and functionality are influenced by diverse cytokines, alone, or in synergy. We observed increased cytokine levels in the sera of wild-type mice, but not in the sera of NKT-cell deficient Jα281−/− mice after αGalCerMPEG administration confirming their dependence on NKT-cell activation (Supporting Information Fig. 2A). NKT-cell characteristic cytokines were predominantly detected 6 h after treatment (e.g. IFNγ, IL-4), whereas a more diverse cytokine environment was observed after 24 h (IFNγ, IL-2, IL-12, IL-15, IL21, GM-CSF, and TNFα) (Supporting Information Fig. 2B). The observed changes in the cytokine profile suggest the generation of an NK-cell polarizing microenvironment. Thus, the induced cytokine milieu indicates the stimulation of functionally active NKcell subsets due to cytokines produced by αGalCerMPEG-activated NKT cells.

Activated NKT cells imprint NK-cell differentiation and functionality To address the impact of αGalCerMPEG-activated NKT cells on NK-cell differentiation, experiments in wild type (C57BL/6) and Jα281−/− mice were performed. At steady state, the distribution of NK-cell subsets does not differ in wild type and Jα281−/− mice. Following treatment, the increased frequency of the CD27high Mac-1low and CD27high Mac-1high NK-cell subsets detected in wildtype mice was not observed in Jα281−/− mice (Fig. 2A). This www.eji-journal.eu

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Figure 1. Stimulation with the NKT-cell agonist αGalCerMPEG induces changes in NK-cell numbers, proliferative capacity, and differentiation status. Splenocytes were isolated from C57BL/6 mice at the indicated time points after a single dose s.c. injection of αGalCerMPEG (3.4 nmol), cells were stained for NK-cell markers (NKp46+ CD3− ) and further analyzed by FACS. For in vitro experiments sorted NK cells were coincubated with bone marrow-derived DC and sorted NKT cells in the presence of 300 ng/mL αGalCerMPEG for 24 h. (A) Absolute lymphocyte numbers and (B) absolute NK-cell numbers were determined by flow cytometry (n = 10–11). Box plots represent the data’s range of variation (minimum to maximum) and vertical lines show the mean value. (C) Absolute cell numbers of CD27 and Mac-1 expressing NK cells were determined by flow cytometry (n = 13). (D) Representative FACS staining for CD27 and Mac-1 expressing NK cells. One representative plot out of at least three independent experiments is shown. (E) Relative distribution of CD27 and Mac-1 expressing NK cells evaluated after in vivo and (F) in vitro stimulation with αGalCerMPEG (in vivo n = 13–18, in vitro n = 15 mice). Mice were injected with αGalCerMPEG and then treated with 1 mg BrdU i.p. daily. Splenocytes were collected 72 h after injection and stained for NK cells, CD3+ cells and BrdU incorporation. (G) Frequency of proliferating cells (n = 3 – 6 mice). (Left) Bars show mean ± SEM of data from one out of two independent experiments. (Right). Histograms display a representative staining for BrdU incorporation by NK and CD3+ cells (filled grey = untreated, black line = treated). (H) Relative distribution of CD27 and Mac-1 expressing cells within BrdU+ NK cells (n = 10 mice). (C, E, F and H) Bars. Columns represent the mean ± SEM of data pooled from two or more independent experiments. Asterisks denote significant values as calculated by One-way or Two-way ANOVA as compared to untreated controls or the NK-cell subset with the highest abundance (H); **** p  0.0001; *** p  0.001; * p  0.05; n.s., not significant.

strongly suggests that αGalCerMPEG-activated NKT cells affect the differentiation status of NK cells. The increased frequency of CD27high NK cells in wild-type mice indicates an improved NKcell functionality. Therefore, it was assessed if αGalCerMPEGmediated stimulation of NKT cells influences NK-cell activation,  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

cytokine secretion, and cytotoxic activity. To this end, splenic NK cells obtained 72 h after αGalCerMPEG administration were coincubated with YAC-1 target cells and further analyzed for the expression of the activation marker CD69, IFNγ secretion, and the expression of the degranulation marker CD107a. A www.eji-journal.eu

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significantly increased percentage of functional NK cells and enhanced expression of CD69, IFNγ, and CD107a were observed in splenocytes isolated from αGalCerMPEG-treated mice (Fig. 2B, C, H). These effects were also observed in mice treated with the parental compound αGalCer, but the pegylated derivative exhibited considerably stronger biological activities (Supporting Information Fig. 3). In vitro studies in which bone marrow-derived DCs pulsed with αGalCerMPEG were cocultured with spleen-derived NKT and NK cells further supported the improved NK-cell functionality (Fig. 2D, E). To further evaluate the dependence of the observed NK-cell activation on NKT cells, experiments were performed using Jα281−/− mice. The obtained data support a key role for activated NKT cells, since no major changes were observed with respect to CD69 expression, IFNγ secretion, and degranulation activity in Jα281−/− mice following αGalCerMPEG administration (Fig. 2F, G, I). To address if the higher differentiation status induced by activated NKT cells is responsible for the improved functionality, CD69+ , IFNγ+ , or CD107a+ splenic NK cells derived from αGalCerMPEG-treated wild-type mice were evaluated with respect to their expression of CD27 and Mac-1. CD69+ NK cells displayed a distribution over all four subsets. IFNγ+ and CD107a+ NK cells, analyzed following coincubation with the NK-cell sensitive target cell line YAC-1, were mostly found to be CD27high Mac-1high (Fig. 2J). This is consistent with the described higher frequency of the CD27high NK-cell subsets detected in the spleen after αGalCerMPEG administration. The obtained data indicate that the increased percentage of highly functional CD27high Mac1high NK cells observed following NKT-cell activation is accompanied by improved NK-cell activation, cytokine secretion, and killing capacity.

Changes in the expression level of the NK-cell transcription factors Eomes and T-bet To assess whether activation of NKT cells affects transcription factors relevant for NK-cell maturation and functionality, the expression of Eomes and T-bet was evaluated. A significantly increased expression of Eomes on NK cells was observed 72 h after αGalCerMPEG stimulation (Fig. 3A). The analysis of Eomes expressing NK cells with regard to their expression of CD27 and Mac-1 showed an increased relative abundance of CD27high

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NK cells within Eomes+ NK cells following NKT-cell activation as compared to the untreated control (Fig. 3B). T-bet expression on NK cells was diminished after NKT-cell activation (Fig. 3C). However, T-bet expressing NK cells were mainly found to be CD27high in the αGalCerMPEG-treated group as compared to the untreated control (Fig. 3D). Thus, NKT-cell activation led to changes in the expression level of transcription factors crucial for NK-cell differentiation and functionality, especially of Eomes.

Educated NK cells represent the main responders after NKT-cell stimulation The ability of NK cells to become functionally active is closely connected to their education status. Thus, the results described above raised the question whether the observed NK-cell functionality induced by activated NKT cells is dependent on the education status. NK cells in C57BL/6 (H-2b ) mice are well characterized with respect to their education status [26]. We assessed whether uneducated and educated NK cells, defined by Ly49C and NKG2A expression, display the same distribution pattern with respect to their differentiation status following NKT-cell activation. The αGalCerMPEG-mediated stimulation of NKT cells resulted in significantly higher frequencies of the CD27high Mac-1high NK-cell subset as compared to cells at steady-state conditions, regardless of their education status (Fig. 4A, B). Educated NK cells additionally displayed a significant increase of the CD27high Mac-1low NK-cell subset (Fig. 4B). These changes were not observed in Jα281−/− mice (Fig. 4C, D), thereby suggesting that they are dependent on NKT-cell activation. To further evaluate if the changes in NK-cell functionality induced by NKT-cell activation depend on the education status, splenocytes collected after αGalCerMPEG injection were analyzed with respect to IFNγ production and CD107a expression following coincubation with YAC-1 target cells. NKT-cell stimulation resulted in significantly elevated frequencies of IFNγ+ and CD107a+ educated NK cells as compared to uneducated NK cells. This seems to indicate that the observed increased NK-cell functionality is mainly due to the activation of educated NK cells (Fig. 4E, F). Thus, in vitro cross-linking studies were performed with splenocytes derived from αGalCerMPEGtreated and untreated mice to confirm these findings. NKT-cell



Figure 2. NKT-cell activation induced changes on NK-cell differentiation and functionality. C57BL/6 and Jα281−/− mice were injected by s.c. route with a single dose of αGalCerMPEG (3.4 nmol) and splenocytes were collected 72 h after injection. Cells were stained for NK cells (NKp46+ CD3− ) and dissected into subsets as defined by the expression of CD27 and Mac-1. For in vitro experiments sorted NK cells were coincubated with bone marrow-derived DCs and sorted NKT cells in the presence of 300 ng/mL αGalCerMPEG for 24 h. (A) Relative distribution of CD27 and Mac-1 expressing NK cells at steady state and 72 h after injection of αGalCerMPEG (n = 18 for C57BL/6 mice, n = 11 for Jα281−/− mice). Frequencies display data pooled from three or more independent experiments. Splenocytes were stained for NK cells expressing CD69, CD107a, and IFNγ. (B, C) median fluorescence intensity (MFI) and frequencies of NK cells expressing CD69, IFNγ, and CD107a in C57BL/6 mice in vivo (MFI n = 3 mice, frequencies n = 11–13 mice) and (D, E) in vitro (MFI n = 6 mice, frequencies n = 15 mice). Frequencies display data pooled from three or more independent experiments. (F, G) MFI and frequencies of NK cells expressing CD69, IFNγ, and CD107a in Jα281−/− mice in vivo (MFI n = 3, frequencies n = 10–11 mice). Frequencies display data pooled from two or more independent experiments. (H, I) Representative staining for CD69, IFNγ, and CD107a in C57BL/6 and in Jα281−/− mice. Expression of IFNγ and CD107a were analyzed following 6 h coincubation with YAC-1 target cells. (J) Relative distribution of NK cells derived from C57BL/6 mice expressing CD69 (n = 11 mice), secreting IFNγ (n = 14 mice), and expressing CD107a (n = 17 mice) after treatment. (A–G) Bars represent the mean ± SEM. MFI are representative from one out of at least three independent experiments. Asterisks denote significant values as calculated by unpaired, two-tailed Student’s t-test or One-way ANOVA as compared to untreated samples (B and C) or the NK-cell subset with the highest abundance (F); **** p  0.0001; *** p  0.001; ** p  0.01; n.s., not significant.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 3. Changes in the expression levels of the transcription factors Eomes and T-bet. C57BL/6 mice were injected by s.c. route with a single dose of αGalCerMPEG (3.4 nmol) and splenocytes were collected 72 h after injection. Cells were stained for NK cells (NKp46+ CD3− ), the transcription factors Eomes and T-bet and dissected into subsets as defined by the expression of CD27 and Mac-1. (A) Frequency and MFI of Eomes expression by NK cells. (Left) Frequencies; (Right) Representative histogram. (B) Relative distribution of Eomes+ NK cells expressing CD27 and Mac-1. (C) Frequency and MFI of T-bet expression by NK cells. (Left) Frequencies; (Right) Representative histogram. (D) Relative distribution of T-bet+ NK cells expressing CD27 and Mac-1. The histograms display representative stainings for Eomes and T-bet including unstained samples (filled light grey), fluorescence minus one (FMO, dotted line), untreated samples (filled dark grey), and treated samples (black line). (A–D) Columns represent the mean ± SEM. MFI are representative from one out of two independent experiments (n = 4–5 mice), frequencies display data pooled from two independent experiments (n = 9 mice). Asterisks denote significant values as calculated by unpaired, two-tailed Student’s t-test or one-way ANOVA as compared to untreated samples; **** p  0.0001; *** p  0.001; ** p  0.01; ** p  0.05; n.s., not significant.

activation resulted in the generation of NK cells with a higher responsiveness to anti-NK1.1 stimulation as compared to the untreated group. In this context, educated NK cells were the main responders after anti-NK1.1 stimulation as compared to uneducated NK cells (Fig. 4G, H). The stimulation with anti-CD8, used as negative control, did not result in the activation of NK cells (data not shown). Thus, our data suggests a superior overall functionality of the educated NK-cell subset induced by activated NKT cells.

High CD27high NK-cell frequency following NKT-cell stimulation correlates with increased anti-viral activity Virus-infected cells represent a main target of NK cells. Thus, using a mCMV infection model it was evaluated whether the observed phenotypic changes have functional implications during a viral infection. Treatment of C57BL/6 and BALB/c mice with the NKTcell agonist αGalCerMPEG 24 h before infection resulted in significantly decreased splenic viral titres 72 h postinfection as compared to the untreated control group. NK-cell depletion resulted in a reduced anti-viral activity, thereby confirming the importance of αGalCerMPEG-dependent NK-cell activation in the observed  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

improved viral control (Fig. 5A, B). The importance of NKT cells for the αGalCerMPEG-mediated anti-viral activity was demonstrated by the observation that the posttreatment enhanced viral control was abolished in Rag1−/− and Jα281−/− mice infected with mCMV (Fig. 5C, D). Thus, preactivation of NKT cells with αGalCerMPEG leads to an improved anti-viral immune response, which is at least in part mediated by NK cells. The assessment of the differentiation status of NK cells revealed that untreated but mCMV-infected mice were mainly enriched for the CD27high Mac-1high subset (40%) as compared to steady-state conditions (25%). Treatment prior to infection did not result in a further increase of this subset. However, αGalCerMPEG treatment led to increased frequencies of CD27high Mac-1low NK cells (25%) as compared to untreated (12%) and infected (7%) mice (Fig. 5E). Treatment with αGalCerMPEG prior to infection also resulted in an increased frequency of functional NK cells and enhanced expression of IFNγ and CD107a as compared to cells from mCMV-infected untreated mice (Fig. 5F, G). These changes seem to be dependent on activated NKT cells since Jα281−/− mice displayed only differences due to mCMV infection, but not as a result of αGalCerMPEG treatment (Fig. 5H, I, J). Most IFNγ producing and degranulating NK cells were found to be part of the CD27high Mac-1low and CD27high Mac-1high subsets following NKT-cell stimulation prior to infection, whereas in untreated www.eji-journal.eu

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Figure 4. Educated NK cells represent the main responder after NKT-cell stimulation. C57BL/6 and Jα281−/− mice were injected by s.c. route with a single dose of αGalCerMPEG (3.4 nmol). Splenocytes were collected 72 h later, stained for NK cells (NKp46+ CD3− ), and analyzed for the relative distribution of educated (Ly49C/I+ or NKG2A+ ) and uneducated (Ly49C/I− and NKG2A− ) NK cells, as well as for the expression of IFNγ and CD107a. Uneducated and educated cells were also analyzed for their differentiation status, as defined by the expression of CD27 and Mac-1. (A) Relative distribution of uneducated and (B) educated NK cells derived from C57BL/6 mice (n = 13 mice, pooled data from three independent experiments). (C) Relative distribution of uneducated and (D) educated NK cells derived from Jα281−/− mice (n = 8 mice, pooled data from two independent experiments). (E) MFI and (F) frequencies of IFNγ and CD107a expressed by NK cells following 6 h coincubation with YAC-1 target cells. (G) MFI and (H) frequencies of IFNγ and CD107a expressed by NK cells following 4 h stimulation with immobilized anti NK1.1 antibody. MFI are representative for one out of two independent experiments (n = 4 mice), frequencies display pooled data from two independent experiments (n = 8 mice). Columns represent the mean ± SEM of the data and asterisks denote significant values as calculated by Two-way ANOVA as compared to uneducated cells and comparing untreated and treated educated cells; **** p  0.0001; *** p  0.001; ** p  0.01; **** p  0.05; n.s., not significant.

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Figure 5. Elevated frequency of CD27high NK cells is accompanied by increased antiviral activity. For the determination of the viral load mice were treated with a single dose of αGalCerMPEG (3.4 nmol) by s.c. route 24 h before infection with mCMV. Splenocytes were collected 72 h after infection. Viral loads of splenocytes were assessed and expressed as plaque forming units (PFU). (A) PFU of Balb/c mice after treatment with anti-asialoGM1 and infected with 2 × 105 PFU of mCMV (n = 5 mice). (B) PFU of C57BL/6 mice after treatment with anti-asialoGM1 and infected with 1 × 106 PFU of mCMV (n = 6 mice). (C) PFU of BALB/c Rag1−/− mice infected with 2 × 105 PFU of mCMV (n = 5). (D) PFU of NKT-cell deficient Jα281−/− mice infected with 1 × 106 PFU mCMV (n = 6 mice). Results are presented as scatter dot plots of each single animal; horizontal dashed lines indicate the detection limit of the assay. For the functional analysis, mice were treated s.c. with a single dose of αGalCerMPEG (3.4 nmol) 72 h before infection with mCMV. Splenocytes were collected 24 h after infection. (E, F, G) Cells isolated from C57BL/6 mice and (H, I, J) from Jα281−/− mice were stained for NK cells (NKp46+ CD3− ) and analysed ex vivo for the expression of CD27, Mac-1, IFNγ, and CD107a. (E) Relative distribution of NK-cell subsets as characterised by CD27 and Mac-1 (n = 15 mice). (F) MFI and (G) frequencies of IFNγ and CD107a expressing NK cells (MFI n = 6 mice, frequencies, n = 8–12 mice). (H) Relative distribution of NK-cell subsets characterised by CD27 and Mac-1 derived from Jα281−/− mice. (I) MFI (n = 6) and (J) Frequencies of IFNγ and CD107a expressing NK cells derived from Jα281−/− mice (MFI n = 6 mice, frequencies n = 12 mice). (K) Representative staining of NK-cell subsets within the population of IFNγ secreting and CD107a expressing NK cells (light grey = mCMV, dark grey = mCMV+ αGalCerMPEG treatment). Columns represent the mean ± SEM and display the pooled data of two (Jα281−/− ) and three (C57BL/6) independent experiments for the shown frequencies or representative data of one out of two or more independent experiments for MFI. Asterisks denote significant values as calculated by unpaired Student’s t-test or One-way ANOVA comparing αGalCerMPEG-treated and untreated mCMV-infected mice; **** p  0.0001; *** p  0.001; ** p  0.01; * p  0.05; n.s., not significant.

and mCMV-infected mice, IFNγ+ , and CD107a+ NK cells mainly belong to the CD27high Mac-1high subset (Fig. 5K). The increased frequency of functional CD27high NK cells suggests a correlation between this subset and the observed improved anti-viral NK-cell activity. This hypothesis was supported by in vitro mCMV studies.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Sorted CD27high Mac-1low/high and CD27low Mac-1high NK-cell subsets were coincubated with mCMV-infected cells. The results indicate an improved control of viral growth by CD27high Mac-1low/high NK cells as compared to CD27low Mac-1high NK cells (Supporting Information Fig. 4). www.eji-journal.eu

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Figure 6. Uneducated and educated NK cells are involved in activated NKT-cell-mediated viral control. C57BL/6 mice were injected by s.c. route with αGalCerMPEG (3.4 nmol) and infected with mCMV 72 h later. Splenocytes were collected 24 h after infection, stained for NK cells (NKp46+ CD3− ) and analyzed ex vivo for their education status based on the expression of Ly49C/I and NKG2A, their differentiation status based on the expression of CD27 and Mac-1, and their functionality (IFNγ, CD107a). (A) Relative distribution of uneducated and (B) educated NK-cell subsets expressing CD27 and Mac-1 (n = 15 mice). (C, D) MFI and (E, F) frequencies of IFNγ and CD107a expressing uneducated and educated NK cells (MFI n = 6 mice, frequencies n = 15 mice). Columns represent the mean ± SEM. MFI displays representative data from one out of three independent experiments and frequencies show pooled data from three independent experiments. Asterisks denote significant values as calculated by One-way ANOVA comparing αGalCerMPEG-treated and untreated mCMV-infected mice; **** p  0.0001; ** p  0. 01; * p  0.05; n.s., not significant.

Uneducated and educated NK cells are crucial players for viral control We demonstrated that activated NKT cells induce modifications in NK-cell differentiation, functionality, and education. To assess whether these changes have an effect in mCMV infection, NKcell differentiation based on the education status was analyzed. The differentiation status of uneducated and educated NK cells was influenced by mCMV infection, with enhanced frequencies of CD27high Mac-1high NK cells detected in both groups. Stimulation of NKT cells prior to infection did not result in additional changes in the differentiation of uneducated NK cells as com C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

pared to cells from mice receiving only the mCMV (Fig. 6A). However, αGalCerMPEG-mediated NKT-cell stimulation resulted in an additional increment of both CD27high Mac-1high and CD27high Mac-1low cells in the educated NK-cell subset (Fig. 6B). Interestingly, the analysis of functionally active uneducated and educated NK cells following mCMV infection with prior αGalCerMPEG treatment indicated that production of IFNγ and cytotoxic activity is induced regardless of their education status (Fig. 6C–F). Taken together, these data suggest that the observed anti-viral activity, which is in part mediated by NK cells, is likely due to the activation of both functional uneducated and educated NK cells. www.eji-journal.eu

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Discussion NK-cell responsiveness can be regulated by either direct or indirect interactions with other immune cells [27, 28]. In this context, CD1d-restricted NKT cells stimulated with αGalCer are able to enhance NK cells proliferative capacity, IFNγ production and killing capacity of transformed or viral-infected cells [22, 29]. This renders the NKT-NK-cell interaction an interesting target for clinical applications, which also offers an opportunity to fine-tune potential immune interventions by tailoring them according to the distinct functional properties of the recently identified NK-cell subsets [9, 30]. We observed that treatment with the NKT-cell agonist αGalCerMPEG resulted in elevated levels of highly active CD27high NK cells exhibiting enhanced IFNγ production and killing activity as compared to steady state conditions. In vitro studies performed with bone marrow-derived DCs pulsed with αGalCerMPEG and cocultured with splenic NKT cells confirmed the increased expression of CD27 and Mac-1 on splenic NK cells and their improved functionality. Adoptive transfer studies demonstrated that differentiation of NK cells is a sequential process [8]. Thus, the increased frequencies of CD27high splenic NK cells observed in vivo might result from either local proliferation or migration of immature NK cells from other tissues. We observed that bone marrow NK cells were exclusively found to be CD27high Mac-1low 72 h after αGalCerMPEG-dependent NKT-cell stimulation (data not shown). This is in agreement with previous reports that demonstrate that the bone marrow harbours a high frequency of CD27high Mac-1low NK cells [31]. Thus, the increased frequency of CD27high splenic NK cells might in part result from the migration of bone marrowderived CD27high NK cells. However, it cannot be excluded that terminally differentiated CD27low Mac-1high NK cells might become reactivated in a cytokine-mediated process and reacquire CD27. This is supported by our in vitro coculture studies with DCs, NKT cells, and NK cells in the presence of αGalCerMPEG (Fig. 2D, E). This might further explain the observed decreased frequency of CD27low Mac-1high NK cells following treatment. However, emigration or cell death could also contribute to this observation. Thus, the increased frequency of CD27high NK cells and the decreased frequency of CD27low Mac-1high NK cells observed in vivo are probably due to several biological processes, including migration of immature NK cells, proliferation at local and distant sites, and reactivation of the CD27low Mac-1high NK-cell subset. Changes in the cytokine milieu induced by activated NKT cells are possibly a main driving force for the observed effects. In fact, cytokines known to be involved in NK-cell activation, proliferation, maturation, and survival (e.g. IFNγ, IL-2, IL-12, and IL-15) were elevated after NKT-cell stimulation [32–36]. IFNγ is the most prominent cytokine after NKT-cell activation and several reports demonstrated its importance in the anti-tumor and anti-viral responses of NK cells [35, 37]. Experiments in IFNγR−/− mice showed an increased number of CD27high NK cells as compared to wild-type mice at steady state. Stimulation with αGalCerMPEG resulted in further elevated frequencies of CD27high

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NK cells in both mouse strains (Supporting Information Fig. 5A). However, the observed increase of CD27high NK cells was lower in IFNγR−/− than in wild-type mice. Thus, the pathways induced by IFNγ seem to play a minor role in the generation of the CD27high NK-cell subsets. NKT-cell-mediated NK-cell degranulation capacity seems to be IFNγ independent as no differences were detected between wild type and IFNγR−/− mice (Supporting Information Fig. 5A, B). Several studies have also demonstrated that NKT-cell stimulation results in DC maturation, thereby suggesting an impact of DC activation on NK-cell differentiation and functionality [38, 39]. Studies performed in CD11cDTR mice revealed that at steady state the differentiation of NK cells was not altered by DC depletion as compared to DC competent mice (Supporting Information Fig. 5C). Stimulation of NKT cells led to an increase in CD27high NK cells in both DC depleted and competent mice. However, NKTcell-mediated induction of IFNγ secreting NK cells was impaired in DC depleted mice (Supporting Information Fig. 5C, D). This suggests that NKT-cell-dependent generation of IFNγ-producing NK cells requires the help of DCs, whereas differentiation and cytotoxic activity occurs independently. Recent reports demonstrated the importance of DC-derived IL-15 and DC-NK-cell-to-cell contact for the induction of NK-cell-derived IFNγ [40, 41]. Thus, the observed DC-dependency of IFNγ production by NK cells after NKT-cell activation suggests that IL-15 might be involved in this process. However, in vitro IL-15 blocking experiments could not confirm the importance of IL-15 in the NKT-cell-mediated modulation of NK cells (Supporting Information Fig. 5E, F). Therefore, the observed changes are most probably the result of synergistic effects mediated by diverse activating cytokines. In this regard, IL12 was also detected in sera after αGalCerMPEG stimulation and might be relevant for NKT-cell-induced NK-cell differentiation and functionality [42]. The transcriptional pathways leading to NK-cell activation are mainly mediated by the transcription factors Eomes and T-bet via IL-12, IL-15, or IL-18 activation [43]. The observed NKT-cellmediated increase in the expression of Eomes might contribute to explain the enhanced secretion of IFNγ. The αGalCerMPEGinduced reduction of T-bet expression might explain the observed accumulation of CD27high NK cells. In fact, previous studies showed that T-bet is involved in the suppression of CD27 expression [44]. It is considered that educated NK cells can become functionally competent [45]. However, NKT-cell-mediated effects on the distribution of uneducated and educated NK cells with respect to their differentiation status have not been assessed. We observed a higher frequency of CD27high cells among uneducated NK cells, as compared to the educated ones. This indicates that at steady state a high proportion of uneducated NK cells display a lower activation threshold. NKT-cell activation induced changes in the differentiation of both uneducated and educated NK cells, albeit to a different extent. The findings that mainly educated NK cells displayed elevated IFNγ secretion and high degranulating capacity evidence a crucial role of activated NKT cells in the generation

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of highly functional NK cells. This is in accordance with published studies on human and murine NK cells, where mainly educated NK cells displayed functional capacities [16, 46]. Although uneducated NK cells are considered to be hyporesponsive to ensure self-tolerance, they were described to be responsible for mCMV clearance [47]. The results obtained here contribute toward the understanding of this apparent dilemma. We detected a higher frequency of both, uneducated and educated CD27high Mac-1high NK cells 24 h after infection. A higher frequency of uneducated NK cells being CD27high Mac-1high (50%) was observed as compared to the educated subset (40%). This indicates that uneducated NK cells are more prone to become activated in the course of mCMV infection. However, we could not observe differences in NK-cell functionality depending on the education status when samples were evaluated 24 h after infection. On the other hand, NKT-cell stimulation prior to mCMV infection resulted in elevated frequencies of the CD27high Mac-1low and CD27high Mac-1high NK-cell subsets, especially among educated NK cells as compared to infected but untreated mice. Interestingly, the functionality of NK cells seems to be independent of the education status in this in vivo viral infection model. This is in contrast to the data obtained by the in vitro YAC-1 killing and cross-linking studies (Fig. 4), which clearly demonstrated the functional superiority of the educated NK cells induced by NKT-cell stimulation. These apparently contrasting observations might be explained by a combined NK-cell activation as a result of both mCMV infection and NKT-cell stimulation. NK-cell education and differentiation are most probably uncoupled processes that are differentially influenced by mCMV infection and αGalCerMPEG treatment, which in turn might affect diverse pathways. It still remains to be proven whether the functional CD27high NK cells induced by NKT cells are the main cells responsible for the observed anti-viral response. Preliminary in vitro experiments suggest that CD27high NK cells controlled viral growth better than CD27low Mac-1high NK cells and therefore seem to be important players in mCMV viral growth control (Supporting Information Fig. 4). This is consistent with recent findings underscoring the importance of CD27 for the generation of T cell immunity against mCMV [48]. However, it cannot be ruled out that other soluble mediators or effector cells could also contribute to the overall anti-viral activity. In conclusion, our findings suggest that crucial NK-cell characteristics can be effectively manipulated by the stimulation of NKT cells. The generation of highly active NK cell subsets renders this approach particularly attractive for clinical applications. NK cells are already used as a valid target in cancer therapy [49, 50]. However, it is becoming clear that NK cells could also be exploited as a target for immune intervention against infections [51]. In this regard, several reports described the appearance of antigenspecific memory-like NK cells that are detectable over long time periods [52, 53]. Future studies should dissect the cross-talk of NK cells with other components of the immune system, evaluating their impact in the initiation and maintenance of antigen-specific adaptive immunity.

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Material and methods Mice BALB/c (H-2d ) and C57BL/6 (H-2b ) mice were purchased from Harlan Winkelmann GmbH (Borchen, Germany). Jα281−/− mice (C57BL/6 background), Rag1−/− , and CD11cDTR mice (BALB/c background) were bred at the animal facility of the Helmholtz Centre for Infection Research, Braunschweig. Mice were treated in accordance with local and European Community guidelines and kept under specific pathogen-free conditions in individual ventilated cages with food and water ad libitum. The animal experiments have been approved by the local government in Braunschweig (Germany) under the animal permission codes 33.9– 42505–04–12/0942 and 33.42502–13/1305. IFNγR−/− mice were bred and housed under standard conditions at the Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Stockholm. All procedures were performed under both institutional and national guidelines.

Synthesis of αGalCerMPEG To produce water soluble αGalCer, a pegylated derivative was generated as described in Ebensen et al. [25]. Briefly, the parental compound αGalCer was mixed with methyl-PEG-COOH. The resulting compound αGalCerMPEG was purified by silica gel chromatography and the purity was analyzed by HPLC.

In vivo assessment of NK-cell activation by αGalCerMPEG To study the in vivo effect of αGalCerMPEG on NK-cell activation and functionality, C57BL/6 (H-2b ) mice received 3.4 nmol αGalCerMPEG by s.c. route. Control mice were treated with PBS. At different time points single cell suspensions derived from spleens were prepared and analyzed by multicolour flow cytometry for activation makers, transcription factors, intracellular cytokine production, and degranulation.

Preparation of single cell suspension and flow cytometry analysis of murine NK cells Spleens were prepared and treated as single samples throughout the experiment. Single cell suspensions were obtained by mincing the organ through a 100 μm cell mesh. To destroy erythrocytes, splenocytes were resuspended in ammonium chloride potassium (ACK) lysis buffer. Single cell suspensions were incubated with FcR-block, followed by the surface marker staining. For intracellular and intranuclear staining, cells were fixed and permeabilized with fixation and permeabilization

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solutions (BD Bioscience, USA/eBioscience) according to the manufacturer’s protocol. FACS analysis was performed by FACS LSRII and Fortessa (BD Bioscience, USA) and FlowJo (TreeStar Inc). The following antibodies were used for FACS staining: CD3 (500A2, V500, BD Horizon; 145–2C11, PE, eBioscience), NKp46 (29A1.4, eFluor660, eBioscience), Mac-1 (M1/70, Pacific Blue, eBioscience/ FITC, BD/ Brilliant Violet 421, BioLegend/ V500, BD Horizon), CD27 (LG.7F9, PE-Cy7, eBioscience/PerCP-eF710, eBioscience), CD69 (H1.2F3, PE, BD/ Brilliant Violet 605, BioLegend), IFNγ (XMG1.2, PE/ PerCP-Cy5.5, eBioscience/Brilliant Violet 785, BioLegend), CD107a (eBio1D4B, FITC, eBioscience/APC-Cy7, BioLegend/Brilliant Violet 421, BioLegend), Eomes (Dan11mag, AlexaFluor488, eBioscience), T-bet (4B10, PE-Cy7, BioLegend), Ly49C/I (5E6, PE, BD Pharmingen), NKG2A/C/E (20d5, FITC, eBioscience). To determine viability, the LIVE/DEAD Fixable Blue Dead cell stain kit (UV excitation, Invitrogen) was used. NK cells were gated as displayed in the gating strategy (Supporting Information Fig. 5).

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Cytokine detection in sera after αGalCerMPEG stimulation Cytokine secretion in sera of αGalCerMPEG-treated mice was detected using a bead-based flow cytometry kit (Affymetrix/ eBioscience) according to the supplier’s protocol. Samples were analyzed using FACS Fortessa (BD Bioscience, USA) and FlowCytomixTM Pro 3.0 Software (Affymetrix/eBioscience).

In vivo BrdU uptake C57BL/6 (H-2b ) mice were injected by s.c. route with 3.4 nmol of αGalCerMPEG or PBS followed by injection with 1 mg BrdU i.p. daily. BrdU incorporation was analyzed by flow cytometry 72 h after treatment (BrdU Flow Kit, BD Pharmingen) using FACS LSRII (BD Bioscience, USA) and FlowJo (TreeStar Inc.).

Degranulation and cross-linking assays mCMV infection Both assays were performed with isolated splenocytes as effector cells derived from mice treated with αGalCerMPEG or PBS. For the degranulation assay effector cells were coincubated with the murine NK target cell line YAC-1 at an E:T ratio of 10:1 and with anti-CD107a antibody for 6 h. For the cross-linking assay, plates were coated with purified antibodies for 1 h at 37°C followed by overnight incubation at 4°C. A total of 1 × 106 splenocytes/well were added and incubated for 4 h. After 1 h 5 μg/mL monensin and brefeldin A (Sigma) were added to prevent internalization of CD107a and cytokine release in both assays. The following purified antibodies were used: NK1.1 (PK136, eBioscience/Affymetrix), CD8a (53–6.7, eBioscience).

Mice were treated with αGalCerMPEG or PBS and infected 24 or 72 h later with wild-type mCMV (Smith strain repair, pSM3frMCK-2fl) by i.p. route. BALB/c (H-2d ), BALB/c Rag1−/− , and Jα281−/− mice were infected with 2 × 105 PFU and C57BL/6 with 1 × 106 PFU mCMV per mouse. The virus was prepared as previously described [54]. Viral load in spleen was analyzed in a plaque assay 72 h after infection. For NK-cell depletion, mice were treated twice with 80 μg of anti-asialoGM1 antibody (eBioscience) by i.p. route 24 h prior to αGalCerMPEG treatment and at the day of infection. The NK-cell functionality was assessed by flow cytometry 24 h after infection.

Coculture experiments Statistical analysis The in vitro effects of αGalCerMPEG on NK cells were assessed by coculturing αGalCerMPEG pulsed bone marrow-derived DCs with sorted NKT and NK cells. For the generation of bone marrowderived DCs, monocytes isolated from the bone marrow were cultured with 5 ng/mL GM-CSF for 7 days. Following, bone marrow-derived DCs were harvested and primed with 300 ng/mL αGalCerMPEG for 1 h. The FACS Aria II system (BD Bioscience, USA) was used to sort NKT cells (CD11c− B220− CD4+ CD8+ NK1.1+ ) and NK cells (B220− CD11c− CD4− CD8− NK1.1+ NKp46+ ). The following antibodies were used for the staining: NKp46 (29A1.4, eFluor660, eBioscience), CD4 (GK1.5, FITC, eBioscience), CD8 (53–6.7, FITC, BD), NK1.1 (PK136, PE-Cy7, eBioscience), B220 (RA3–6B2, Pacific Blue, BioLegend), CD11c (N418, PB, BioLegend). Bone marrow-derived DCs, NKT cells and NK cells were cocultured at a ratio of 1:4:10 in medium supplemented with 300 ng/mL αGalCerMPEG for 24 h. The analysis of NK-cell phenotype and functionality was performed using FACS analysis as described above.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Data were analyzed using the GraphPad Prism software (GraphPad Software, USA). Statistical differences were assessed as indicated by either using Student’s t-test, One-way ANOVA or two-way ANOVA. Values of p  0.05 were considered significant.

Acknowledgments: These studies were funded by the European Research Training Group 1273 “Strategies of human pathogens to establish acute and chronic infections“(IRTG1273, DFG). We are grateful to Blair Prochnow for critical reading of the manuscript. Further we thank Ulrike Br¨ oder, Carolin Kanzler, and Hanna Shkarlet for their great supportive technical assistance, Thomas Ebensen, Kai Schulze, and Sebastian Weißmann for fruitful scientific discussions and Julia Holzki and Rosaely Casalegno for www.eji-journal.eu

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technical support with the in vitro mCMV studies. CD11cDTR mice were kindly provided by Petra Dersch.

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15 Kim, S., Poursine-Laurent, J., Truscott, S. M., Lybarger, L., Song, Y. J., Yang, L., French, A. R. et al., Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 2005. 436: 709–713.

Conflict of interest: Carlos A. Guzm´ an is named as inventor in a patent application covering the use of αGalCerMPEG as adjuvant (EP 05 022 771.9). Tobias May has filed a patent that covers the technology for establishing conditionally immortalized cell lines.

16 Brodin, P., Karre, K. and Hoglund, P., NK cell education: not an on-off switch but a tunable rheostat. Trends Immunol. 2009. 30: 143–149. 17 Sun, J. C., Beilke, J. N. and Lanier, L. L., Adaptive immune features of natural killer cells. Nature 2009. 457: 557–561. 18 Marcus, A. and Raulet, D. h., Evidence for natural killer cell memory. Curr. Biol. 2013. 23: R817–R820. 19 Paust, S. and von Andrian, U. h., Natural killer cell memory. Nat. Immunol.

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Abbreviations: BrdU: bromodeoxyuridine · αGalCer: α-galactosylceramide · αGalCerMPEG: pegylated derivative of α-galactosylceramide

2252–2258. 42 Borg, C., Jalil, A., Laderach, D., Maruyama, K., Wakasugi, H., Charrier, S., Ryffel, B. et al., NK cell activation by dendritic cells (DCs) requires the formation of a synapse leading to IL-12 polarization in DCs. Blood 2004. 104: 3267–3275. 43 Glimcher, L. h., Townsend, M. J., Sullivan, B. M. and Lord, G. M., Recent

Full correspondence: Dr. Peggy Riese, Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany Fax +4953161814699 e-mail: [email protected]

developments in the transcriptional regulation of cytolytic effector cells. Nat. Rev. Immunol. 2004. 4: 900–911. 44 Gordon, S. M., Chaix, J., Rupp, L. J., Wu, J., Madera, S., Sun, J. C., Lindsten, T. and Reiner, S. L., The transcription factors T-bet and Eomes control key

Current address: Tobias May, InSCREENeX GmbH, Braunschweig, Germany.

checkpoints of natural killer cell maturation. Immunity 2012. 36: 55–67. 45 Hoglund, P. and Brodin, P., Current perspectives of natural killer cell education by MHC class I molecules. Nat. Rev. Immunol. 2010. 10: 724–734. 46 Bjorkstrom, N. K., Riese, P., Heuts, F., Andersson, S., Fauriat, C., Ivarsson, M. A., Bjorklund, A. T. et al., Expression patterns of NKG2A, KIR, and CD57

 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 22/9/2014 Revised: 5/2/2015 Accepted: 19/3/2015 Accepted article online: 23/3/2015

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