- Open Access
Activated regulatory T cells suppress effector NK cell responses by an IL-2-mediated mechanism during an acute retroviral infection
© Littwitz-Salomon et al. 2015
- Received: 24 March 2015
- Accepted: 16 July 2015
- Published: 30 July 2015
It is well established that effector T cell responses are crucial for the control of most virus infections, but they are often tightly controlled by regulatory T cells (Treg) to minimize immunopathology. NK cells also contribute to virus control but it is not known if their antiviral effect is influenced by virus-induced Tregs as well. We therefore analyzed whether antiretroviral NK cell functions are inhibited by Tregs during an acute Friend retrovirus infection of mice.
Selective depletion of Tregs by using the transgenic DEREG mouse model resulted in improved NK cell proliferation, maturation and effector cell differentiation. Suppression of NK cell functions depended on IL-2 consumption by Tregs, which could be overcome by specific NK cell stimulation with an IL-2/anti-IL-2 mAb complex.
The current study demonstrates that virus-induced Tregs indeed inhibit antiviral NK cell responses and describes a targeted immunotherapy that can abrogate the suppression of NK cells by Tregs.
- NK cells
- Friend retrovirus
- Regulatory T cells
- IL-2 consumption
- Treg suppression
Natural killer (NK) cells are innate effector lymphocytes that survey the body and destroy viral pathogens without previous sensitization. Activation of NK cells is mediated by a repertoire of competing activating and inhibitory signals of germline-encoded receptors. This dynamic balance between inhibitory and activating receptors allows NK cells to recognize and eliminate infected or tumor cells while leaving out normal unaltered cells. Activated NK cells produce proinflammatory cytokines such as interferon-γ (IFN-γ) or tumor necrosis factor-α. They can also induce apoptosis in target cells by the expression of cytotoxic molecules like granzymes (Gzm) or perforin or death receptor ligands on their cell surface including tumor necrosis factor related apoptosis inducing ligand (TRAIL) and Fas ligand. Potent activation and proliferation of NK cells depends on Interleukin (IL)-2, originally described as a T cell growth factor [1, 2]. IL-2 influences different lymphocyte subsets during immune responses, differentiation, survival and homeostasis. The IL-2 receptor is comprised of three subunits—alpha (CD25) , beta (CD122) and gamma (CD132) [4–6]. CD122 is broadly expressed on NK cells whereas CD25 is highly expressed on activated T cells, especially on a distinct CD4+ T cell population called regulatory T cells (Tregs) [4–6]. Tregs are crucial for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammation [7, 8]. However, by inhibition of virus-specific effector T cells the suppressive activity of Tregs can also be associated with progressive T cell exhaustion and establishment of chronic viral infections, as shown for human immunodeficiency virus (HIV) or hepatitis C virus infection of humans or the murine Friend retroviral (FV) infection [9–12]. Interestingly, Tregs do not merely suppress T cells but can also inhibit NK cell functions in autoimmune diseases, cancer and pregnancy [13, 14]. Particularly with regard to viral infections, NK cells contribute to the control of HIV-1 through recognition of infected cells by activating and inhibitory killer immunoglobulin-like receptors (KIRs) [15–18]. The important role of NK cells in controlling retroviral infections was demonstrated in studies showing that polymorphisms in KIRs as well as active suppression of ligand expression for the activating natural killer group 2 member D (NKG2D) receptor by HIV-1 resulted in reduced NK cell activity in HIV-1 infection [19, 20]. In our previous work, we also demonstrated an antiretroviral function of murine NK cells during the early phase of infection with FV .
It was recently described that Tregs control a subpopulation of CD127+ NK cells in naive mice . Another study also showed Treg-mediated NK cell suppression during homeostasis, which was not seen during murine cytomegalovirus (CMV) infection . However, to define the influence of Tregs on NK cell responses in viral infections, Tregs have to be experimentally depleted during an ongoing infection that induces potent Treg responses. We used the well-established FV mouse model, in which a strong activation and expansion of non-virus-specific natural Tregs occurs during the acute FV infection . The FV complex consists of a replication-competent but apathogenic Friend murine leukemia virus and a pathogenic but replication-defective spleen focus-forming virus. Resistant mice, such as C57BL/6, are able to recover from acute infection, but develop a persistent infection . During the acute phase of FV infection, antiviral CD8+ T cells are required for the control of viral replication. However, activated Tregs subsequently start to suppress the effector functions of CD8+ T cells, which contribute to the establishment of viral chronicity [9, 26–29].
Here, we investigated the suppressive capacity of virus-induced Tregs on NK cell responses during acute FV infection taking advantage of the transgenic DEREG mouse model in which Tregs can be selectively depleted by diphtheria toxin injection . We identified a strong increase of NK cell maturation, activation and effector functions in Treg-depleted mice. Suppression by Tregs, which was mediated by IL-2 consumption, led to increased viral loads and reduced cytotoxicity of NK cells in vivo. This suppression could be overcome by application of IL-2/anti-IL-2 mAb complex, which specifically stimulated NK cells.
Thus, defined therapies with IL-2, which specifically improve NK cell responses, should be of interest to augment NK cell responses in viral infections or cancer.
NK cell frequencies inversely correlate with viral loads in Treg-depleted mice
NK cells show enhanced activation and effector molecule expression in the absence of Tregs
Treg ablation results in potent target cell killing by NK cells
Tregs deprive NK cells of IL-2 during acute FV infection
IL-2 binds to the high-affinity trimeric IL-2 receptor (CD25, CD122 and CD132) and to the low affinity dimeric IL-2 receptor consisting of CD122 and CD132 . Tregs express preferably the trimeric IL-2 receptor, whereas dimeric receptors are rather associated with CD8+ T cells and NK cells. Treg depletion did not change the proportion of CD122+ NK cells in comparison to non-depleted mice (data not shown) during FV infection. Analysis of the IL-2R α-chain (CD25) also revealed no changes in the expression levels after Treg depletion in uninfected and FV-infected mice (Fig. 4b, c).
Specific IL-2 stimulation of NK cells mimics the effect of Treg depletion on NK cell activation and differentiation and leads to better FV control
We also detected a significant increase in the expression of the early activation marker CD69 (Fig. 5c) and the maturation marker KLRG1 (Fig. 5d) after stimulation with the IL-2/anti-IL-2 mAb complex which were comparable to those seen in Treg-depleted mice. The combination of both treatments (FV+DT+IL-2/anti-IL-2) did not further increase the expression of both markers (Fig. 5c, d).
Next, we analyzed the differentiation state of NK cells after stimulation with IL-2 by expression of the surface molecules CD11b and CD27 (Fig. 5e). We observed a massive increase in CD11b+CD27− terminally differentiated NK cells (32%) compared to control mice (7%), which was also seen after Treg depletion (FV+DT). This effect is not further increased by additional Treg depletion (FV+DT+IL-2/anti-IL-2), suggesting that NK cells are saturated with IL-2 after application of the IL-2/anti-IL-2 mAb complex. Additional depletion of Tregs might increase the level of circulating IL-2, which is not further consumed by stimulated NK cells.
Finally, we wanted to determine whether the improved NK cell activation by IL-2 treatment has an effect on viral loads. Interestingly, specific NK cell stimulation with IL-2 in the presence of Tregs mediated a significant reduction in viral loads (FV+IL-2/anti-IL-2; Fig. 5f), which was comparable to the results seen in Treg-depleted mice (FV+DT; Fig. 5f). In line with our previous results of NK cell activation and effector function (Fig. 5b–e), the combination of Treg depletion and NK cell stimulation did not further decrease viral burden (FV+DT+IL-2/anti-IL-2; Fig. 5f). To confirm that the observed reduction in the viral loads post IL-2 treatment is mediated by NK cells and not by other cells which express the IL-2 receptor, we depleted CD8+ and CD4+ T cells by injection of supernatant fluid containing CD8-specific mAb 169.4 and CD4-specific mAb YTS 191.1. We stimulated NK cells by application of IL-2/anti-IL-2 mAb complex in these T cell depleted mice and determined viral loads at 12 dpi. As expected, after depletion of CD4+ and CD8+ T cells, the viral loads significantly increased (FV+anti-CD4+anti-CD8; Fig. 5f; Additional file 3), as T cells, especially CD8+ T cells, are required for FV control at that time point . An additional NK cell depletion resulted in a further rise of the viral loads (Additional file 3) demonstrating that NK cells are involved in antiviral immunity during acute FV infection. In contrast, specific NK cell stimulation with IL-2 in T cell-depleted mice (FV+anti-CD4+anti-CD8+IL-2/anti-IL-2) led to a more than 13-fold reduction in the viral loads compared to unstimulated T cell-depleted controls (FV+anti-CD4+anti-CD8) showing that IL-2-stimulated NK cells contribute to FV control.
Additionally we injected IL-2/anti-IL-2 mAb complexes (clone JES-1) targeting Tregs to clarify that specific IL-2 stimulation of NK cells is required for FV control. In contrast to the antibody complexes used before, application of the other IL-2 antibody complex specifically activated Tregs (expression of CD43; Additional file 4c) and this did not result in reduction of viral loads (data not shown).
We also analyzed the activation status of Tregs, CD4+ and CD8+ T cells after treatment with IL-2/anti-IL-2 mAb complexes, but we did not detect any effect of IL-2 on T cells compared to FV-infected control mice (Additional file 4a, b). Furthermore, NK cell depletion during IL-2/anti-IL-2 mAb complex application completely abolished IL-2-mediated antiviral immunity (Fig. 5f). This implies that the therapeutic effect of IL-2 was mediated by NK cells and could overcome the suppressive effect of virus-induced Tregs, which led to control of acute FV infection. Here, we describe a new suppressive mechanism of Tregs on NK cell functions during an acute viral infection that is dependent on IL-2 availability or IL-2 consumption by Tregs.
Efficient immune protection against pathogens such as viruses is usually provided by a successful interaction of innate and adaptive immunity. NK cells participate in the rapid control of early viral replication, whereas virus-specific T cells undergo clonal expansion and thus contribute later to antiviral immunity. A potent immune response has to be counter-regulated to prevent immunopathology. This immune control is efficiently mediated by Tregs, which dampen pathogen-specific effector T cell responses. Besides preventing immunopathology, Treg activity can also contribute to inefficient clearance of pathogens and the establishment of chronic viral infections . However, whether Tregs also influence the initial antiviral response of NK cells was not known.
The current study shows for the first time that NK cell responses are suppressed by virus-induced Tregs during an acute retroviral infection. Specific depletion of Tregs during acute FV infection resulted in improved NK cell maturation, differentiation and effector functions (Fig. 2). The suppression of NK cells was mediated by Treg-driven IL-2 consumption, which can be overcome by specific stimulation of NK cells with an IL-2/anti-IL-2 mAb complex in vivo. Others showed a Treg-mediated suppression of NK cell responses in cancer, autoimmunity or during homeostasis, whereas no studies on Treg-mediated NK cell suppression in viral infections existed. Only one recent study using CMV-infected mice analyzed the impact of Treg depletion on NK cell responses. Lindenberg and colleagues  showed that during acute mouse CMV infection Tregs suppress only T cell responses, whereas NK cell responses were not affected by Tregs. However, CMV infection of mice is not a very useful model to study the interaction of Tregs and NK cells because the virus does not efficiently activate or expand Tregs . The effect of virus-induced Tregs on anti-viral NK cell responses can only be investigated in an infection model with strong Treg responses, like the FV mouse model [9, 41]. In a mouse cancer model, CD4+CD25+ Tregs inhibited NKG2D-mediated NK cell cytotoxicity by TGF-β release and subsequent tumor rejection and metastasis inhibition . Similar results were also shown by Ghiringhelli and colleagues  who described suppression of NK cell cytotoxicity by Tregs in a mouse B16 tumor model and human melanoma patients. Our current data and the described studies imply that negative regulation of NK cell responses by Tregs might be a common feature in infections and cancer. Besides cytotoxic T cells, also NK cell cytotoxicity is controlled by Tregs most likely to avoid immunopathology.
Several molecular mechanisms of Treg suppression of NK cells have been discussed. One mechanism used by Tregs to suppress antitumor responses depends on granzyme B and perforin and thus on direct killing of NK cells and CD8+ T cells by Tregs in allogenic B16 and RMA/S tumors . However, in FV-infected mice, no expression of granzymes has been found in activated Tregs , suggesting that other mechanisms of suppression play a more important role. As TGF-β is an important factor in Treg-mediated suppression of NK cells in cancer models [34, 35], we also analyzed the expression of TGF-β in FV-infected and additionally Treg-depleted animals. We did not detect any differences after depletion of Tregs (data not shown), indicating that TGF-β is not involved in the suppression of antiviral NK cell effector functions during FV infection.
In the mouse B16 melanoma model described above, the authors also demonstrated that an in vitro stimulation of PBMC cultures with IL-2 neutralized the Treg-mediated suppression of NK cells suggesting an important role of IL-2 in Treg-NK cell interaction . Our findings and recent studies suggest a cellular interplay of Tregs, CD4+ helper T cells and NK cells. Tregs do not produce IL-2 by themselves, but they consume IL-2 which is mainly produced by CD4+ T cells. The hypothesis is that Tregs dampen the bioavailability of IL-2 by massive consumption of the cytokine or inhibition of IL-2 production by CD4+ T cells and thus indirectly inhibit NK cells due to IL-2 deprivation. There is evidence from a mouse diabetes model showing the importance of Treg suppression and IL-2 consumption in an autoimmune disease . Here, Tregs limited the availability of IL-2 in the pancreatic islets, which dampened activation and effector functions of infiltrating NK cells. However, in the absence of Tregs, IL-2 produced by infiltrating CD4+ helper T cells activated NK cells and enhanced their IFN-γ production which leads to the onset of diabetes. This effect was prevented by application of an IL-2 antagonist in Treg-depleted mice demonstrating the importance of IL-2 in regulating NK cell functions. Others found that depletion of Tregs in Foxp3.DTR mice did not change NK cell tolerance to self- or non-self-ligands, whereas missing-self-induced cytotoxicity of NK cells was enhanced in Treg-depleted mice. They showed that this was dependent on CD4+ helper T cells producing IL-2 and that regulating IL-2 levels may be a mechanism of Tregs to tune NK cell activity . During acute FV infection, IL-2 is mainly produced by CD4+ helper T cells [24, 36], which was subsequently consumed by expanded Tregs. NK cells only received sufficient IL-2 signaling for full activation when the IL-2 consumption by Treg was limited. Thus, our previous data and the current results suggest an interplay of CD4+ helper T cells, Tregs and NK cells also in viral infections with IL-2 being the specific link between these cell populations. Gasteiger et al.  described that Tregs mainly control a very specific NK cell subpopulation (CD127+) during homeostasis, which was also dependent on the availability of IL-2. However, this population of NK cells does not seem to play a major role during FV infection, as it accounted for only 0.5% of all NK cells in infected mice, and this frequency did not change after depletion of virus-induced Tregs.
Immunotherapy with IL-2 is already used in the clinics to treat patients with metastatic melanoma and renal cell carcinoma with objective clinical response rates of 15–25%. As IL-2 has multiple activities on various lymphocytes, treatment with IL-2 results in a variety of different side effects. Many trials have been performed to reduce these by changing the dose, route, or chemical modification of IL-2 or adding additional immunomodulators; however, they were not successful in enhancing the clinical responses . Another attempt is to selectively target distinct cell populations by IL-2 immunotherapy. The IL-2 receptor consists of three subunits: CD25 (α), CD122 (β) and CD132 (γ) . CD25 is highly expressed on Tregs, but hardly expressed on NK cells and memory-phenotype CD8+ T cells . Addition of anti-IL-2 mAb to recombinant IL-2 can target the cytokine to specific cell subsets. In mice, two different clones of anti-IL-2 mAb are available that bind to different sites of the cytokine. Clone JES6-1 shields the CD122 binding site which further targets this antibody to CD25, whereas clone S4B6 interferes with the CD25 binding site of IL-2 and thus leads to preferential CD122 binding [38, 47]. Application of one of these antibodies together with recombinant IL-2 can therefore target IL-2 to Tregs (clone JES6-1) or to NK cells or memory-phenotype CD8+ T cells (clone S4B6). In our study, we used clone S4B6 to specifically stimulate NK cells and reduce binding to Tregs. We observed a significant activation, maturation and differentiation of NK cells upon this IL-2 immunotherapy (Fig. 5), whereas no stimulatory effect was detected on different T cell subsets (Additional file 2). The selective IL-2 stimulation of NK cells reduced viral loads (Fig. 5f) and could overcome suppressive Treg activity suggesting that this might be an interesting new approach for the treatment of infectious diseases. Studies in a B16 melanoma model have shown that immunotherapy with an IL-2/anti-IL-2 mAb complex only binding to the IL-2βγ receptor abrogated IL-2 therapy mediated side effects (pulmonary edema) and led to a vigorous activation of cytotoxic immune cells with strong antitumor reactivity . In their mouse model for autologous hematopoietic stem cell transplantation, Newman and colleagues demonstrated that vaccination with lymphoma cells secreting antibodies in combination with IL-2/anti-IL-2 mAb complexes (clone S4B6) induced strong antitumor responses of cytotoxic cells (CD8+ T cells and NK cells) and thus prevented the establishment of hematologic cancers . Others compared the efficacy of the IL-2/anti-IL-2 mAb complex with IL-2 treatment alone in a B16 melanoma mouse model. The combined treatment could markedly enhance cytotoxicity of CD8+ T cells and partially protect mice from tumor metastasis compared to IL-2 treatment alone . Application of IL-2/anti-IL-2 mAb (clone S4B6) complexes strongly extends the half-life of soluble IL-2 and prevents binding to CD25 on Tregs resulting in a potent improvement of the biological activity of the complex compared to soluble IL-2 . However, these studies did not discuss the role of Tregs in their infection models .
Similar antibodies for IL-2 to target different cell subsets also exist for the use in humans. Complexes of human IL-2 and anti-IL-2-mAB clone MAB602 closely resemble S4B6-IL-2 complexes targeting IL-2 to CD122 on the surface of immune cells. An equivalent for JES6-1 directing IL-2 to CD25 (mAb 5344) does also exist. Also, two mutated isoforms of human IL-2 were developed with increased binding to either CD25 or CD122 [51, 52] implying that targeted IL-2 immunotherapies in humans may be feasible in the future.
To our knowledge, this is the first study showing that immunotherapy with an IL-2/anti-IL-2 mAb complex can overcome Treg suppression and improve NK cell responses in an acute retroviral infection. The specific stimulation of NK cells by IL-2 can result in improved control of viral infections. Such a therapeutic approach has a clear advantage over Treg depletion or manipulation during viral infections, as it may significantly reduce the risk of inducing immunopathologies. Using antibody–cytokine-complexes to specifically target distinct immune cell populations may have a strong impact in future therapies against infections and cancer, as side-effects in patients can be reduced and clinical responses improved.
Natural killer (NK) cells, which belong to the innate arm of the immune system, can contribute to the control of many viral infections. NK cells can directly kill virus-infected cells and thus reduce viral replication. However, in infections with retroviruses, like HIV, NK cells are not sufficient to prevent pathology. Here, we showed that NK cell effector functions are strongly inhibited by virus-induced regulatory T cells leading to failure of NK cells to control retroviral infection. We described IL-2-mediated suppression mechanism by regulatory T cells which can be overcome by NK cell-specific stimulation with IL-2 complexes. This new therapeutic approach to target specific immune cells by antibody–cytokine-complexes might have great potential for the treatment of many viral infections or cancer.
Animal experiments were performed in strict accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). The protocol was approved by the North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection (LANUF) (Permit Number: G1341/12). All efforts were made to minimize suffering.
Mice and virus
Experiments were done using sex- and age-matched inbred C57BL/6 (B6, Harlan Laboratories, Germany) and wild type and transgenic DEREG mice . At the beginning of the experiments, mice were 6–10 weeks old. The FV stock used in these experiments was a FV complex containing B-tropic Friend murine leukemia helper virus and polycythemia-inducing spleen focus-forming virus. The stock was prepared as a 15% spleen cell homogenate from BALB/c mice infected 14 days previously with 3,000 SFFU of FV. Mice were injected intravenously with 0.15 ml phosphate-buffered saline (PBS) containing 20,000 SFFU of FV. The virus stock did not contain lactate dehydrogenase-elevating virus. Mice were sacrificed at 12 dpi by cervical dislocation.
Detection of FV-infected cells in spleen
Infectious centers (IC) were detected by tenfold dilutions of single-cell suspensions onto Mus dunnis cells. Cultures were incubated for 3 days, fixed with ethanol, stained with F-MuLV envelope-specific monoclonal antibody 720 and developed with peroxidase-conjugated goat anti-mouse antibody and aminoethylcarbazol to detect foci .
IL-2 concentration in the serum
For detection of IL-2 concentration in FV-infected mice, sera were harvested at 12 dpi. Cytokine concentration was measured by an IL-2 ELISA (eBioscience) according to the manufacturer’s protocol.
Flow cytometry staining
Cell surface staining was performed using the following antibodies: anti-CD3 (17A2, eBioscience), anti-CD4 (GK1.5, eBioscience), anti-CD8 (53-6.7, eBioscience), anti-CD11b (M1/70, BD Bioscience), anti-CD25 (PC61, BD Bioscience), anti-CD27 (LG.3A10, BioLegend), anti-CD43 (1B11, BioLegend), anti-CD49b (DX5, eBioscience), anti-CD69 (H1.2F3, eBioscience), anti-CD127 (AFR34, eBioscience), anti-KLRG1 (2F1, eBioscience), anti-NK1.1 (PK136, BD Bioscience), anti-TRAIL (N2B2, eBioscience). Dead cells were excluded from analysis via fixable viability dye (eBioscience). For intracellular IFN-γ staining, cells were stimulated with Ionomycin (500 ng/ml), PMA (25 ng/ml) and Brefeldin A (2 μg/ml) and incubated for 3 h at 37°C. For intracellular staining of IFN-γ (XMG1.2, eBioscience) and GzmB (clone GB11, Life technologies), cells were fixed and permeabilized with CytoFix/CytoPerm (BD Bioscience) for 10 min. Intranuclear fixation was performed following the manufacturer’s instruction (Foxp3/Transcription Factor Fixation/Permeabilization, eBioscience). Tregs were stained with antibodies against Foxp3 (FJK-16S, eBioscience) and proliferation was detected using Ki-67 (SolA15, eBioscience). Data were acquired on LSR II flow cytometer (BD Bioscience) and analyses were performed using FACSDiva (BD Bioscience) and Flow Jo (Tree Star, USA) software.
BrdU treatment and staining
5-Bromo-2′-deoxyuridine (BrdU, Sigma) was added to the drinking water of mice and was replaced every day (40 mg). In addition, mice were injected with BrdU every day starting at day 9 (i.p., 1 mg). BrdU staining was performed with BrdU Flow Kit (BD Pharmingen) according to the manufacturer’s protocol. Staining of cells was done with anti-BrdU (Clone BU20A, eBioscience) and acquired on LSR II flow cytometer (BD Bioscience).
IL-2 receptor stimulation
Specific stimulation of NK cells were obtained by intraperitoneal inoculation of 50 µg anti-mouse IL-2 monoclonal antibody (Clone S4B6-1 or JES6-1, BioXCell) and 1 µg of recombinant mouse IL-2 (carrier-free, eBioscience) in 500 µl of PBS. Control groups received 50 µg of isotype control (rat IgG2a, BioXCell). Mice were injected every other day starting from day 7 to day 11 post infection.
NK cell transfer
Splenic NK cells from FV-infected mice were enriched using NK cell isolation Kit II (Miltenyi Biotec) according to the manufacturer’s protocol. Intravenous injection of 2 × 106 NK cells was performed at 10 dpi and viral loads were analyzed 4 days later. Transferred cells contained more than 85% of NK cells and only 2% of T cells.
Depletion of T cells
For depletion of CD8+ T cells mice were injected intraperitoneally with 0.35 ml supernatant fluid containing CD8-specific mAb 169.4 every other day. Intraperitoneal injections of 0.15 ml CD4-specific mAb YTS 191.1 specifically deplete the CD4+ T cell population. At the days of analysis more than 98% of CD4+ T cells and at least 90% of CD8+ T cells were depleted in the spleen. To deplete regulatory T cells transgenic DEREG mice were injected intraperitoneally with DT (0.5 μg, Merck Millipore) diluted in PBS every third day for three times starting at day 5 post infection. The treatment depleted over 97% of CD4+ eGFP+ T cells in the spleen. Wild type DEREG mice injected with DT were used as controls.
NK cell depletion
NK cell depletion was performed as described before . At the day of analysis more than 95% of NK cells (CD3− CD49b+ NK1.1+) were depleted in the spleen.
In vivo NK cell cytotoxicity assay
Statistical analyses and graphical presentations were computed with Graph Pad Prism version 6. Normal distribution was assessed using D’Agostino–Pearson omnibus test. Statistical differences between two different groups were determined by the unpaired student’s t test (parametric distribution) or Mann–Whitney test (non-parametric distribution). Analyses including several groups were tested using Kruskal–Wallis one-way analysis of variance on ranks and Dunn’s multiple comparison test (non-parametric distribution) or the ordinary one-way ANOVA and Bonferroni’s multiple comparisons test (parametric distribution). For the determination of correlating variables, linear regression and Pearson correlation were performed.
ELS designed and performed the experiments, analysed the data, participated in the statistical analysis and wrote the paper. IA, CV and SF performed the experiments. UD conceived and designed the experiments and wrote the paper. KG conceived and designed the experiments, performed the experiments, analysed the data, participated in the statistical analysis and wrote the paper. All authors read and approved the final manuscript.
We thank Adelheid Cerwenka (DKFZ, Heidelberg) for providing us with RMA/S cells. This work was supported by a grant from the University Hospital Essen (IFORES) and by a grant of the Deutsche Forschungsgemeinschaft (TRR 60).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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- Kasaian MT, Leite-Morris KA, Biron CA (1991) The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection. J Immunol 146:1955–1963PubMedGoogle Scholar
- Biron CA, Young HA, Kasaian MT (1990) Interleukin 2-induced proliferation of murine natural killer cells in vivo. J Exp Med 171:173–188PubMedView ArticleGoogle Scholar
- Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, Alt FW (1995) Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521–530PubMedView ArticleGoogle Scholar
- Setoguchi R, Hori S, Takahashi T, Sakaguchi S (2005) Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 201:723–735PubMed CentralPubMedView ArticleGoogle Scholar
- Krieg C, Letourneau S, Pantaleo G, Boyman O (2010) Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells. Proc Natl Acad Sci USA 107:11906–11911PubMed CentralPubMedView ArticleGoogle Scholar
- Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164PubMedGoogle Scholar
- Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N et al (2001) X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 27:18–20PubMedView ArticleGoogle Scholar
- Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M et al (2001) Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 182:18–32PubMedView ArticleGoogle Scholar
- Zelinskyy G, Dietze KK, Husecken YP, Schimmer S, Nair S, Werner T et al (2009) The regulatory T-cell response during acute retroviral infection is locally defined and controls the magnitude and duration of the virus-specific cytotoxic T-cell response. Blood 114:3199–3207PubMedView ArticleGoogle Scholar
- Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF (2004) Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol 78:2454–2459PubMed CentralPubMedView ArticleGoogle Scholar
- Kinter AL, Horak R, Sion M, Riggin L, McNally J, Lin Y et al (2007) CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro. AIDS Res Hum Retroviruses 23:438–450PubMedView ArticleGoogle Scholar
- Cabrera R, Tu Z, Xu Y, Firpi RJ, Rosen HR, Liu C et al (2004) An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection. Hepatology 40:1062–1071PubMedView ArticleGoogle Scholar
- Pedroza-Pacheco I, Madrigal A, Saudemont A (2013) Interaction between natural killer cells and regulatory T cells: perspectives for immunotherapy. Cell Mol Immunol 10:222–229PubMed CentralPubMedView ArticleGoogle Scholar
- Zimmer J, Andres E, Hentges F (2008) NK cells and Treg cells: a fascinating dance cheek to cheek. Eur J Immunol 38:2942–2945PubMedView ArticleGoogle Scholar
- Martin MP, Gao X, Lee J-H, Nelson GW, Detels R, Goedert JJ et al (2002) Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 31:429–434PubMedGoogle Scholar
- Martin MP, Qi Y, Gao X, Yamada E, Martin JN, Pereyra F et al (2007) Innate partnership of HLA-B and KIR3DL1 subtypes against HIV-1. Nat Genet 39:733–740PubMed CentralPubMedView ArticleGoogle Scholar
- Alter G, Teigen N, Ahern R, Streeck H, Meier A, Rosenberg ES et al (2007) Evolution of innate and adaptive effector cell functions during acute HIV-1 infection. J Infect Dis 195:1452–1460PubMedView ArticleGoogle Scholar
- Altfeld M, Goulder P (2007) ‘Unleashed’ natural killers hinder HIV. Nat Genet 39:708–710PubMedView ArticleGoogle Scholar
- Alter G, Heckerman D, Schneidewind A, Fadda L, Kadie CM, Carlson JM et al (2011) HIV-1 adaptation to NK-cell-mediated immune pressure. Nature 476:96–100PubMed CentralPubMedView ArticleGoogle Scholar
- Matusali G, Tchidjou HK, Pontrelli G, Bernardi S, D’Ettorre G, Vullo V et al (2013) Soluble ligands for the NKG2D receptor are released during HIV-1 infection and impair NKG2D expression and cytotoxicity of NK cells. FASEB J Off Publ Fed Am Soc Exp Biol 27:2440–2450Google Scholar
- Littwitz E, Francois S, Dittmer U, Gibbert K (2013) Distinct roles of NK cells in viral immunity during different phases of acute Friend retrovirus infection. Retrovirology 10:127PubMed CentralPubMedView ArticleGoogle Scholar
- Gasteiger G, Hemmers S, Bos PD, Sun JC, Rudensky AY (2013) IL-2-dependent adaptive control of NK cell homeostasis. J Exp Med 210:1179–1187PubMed CentralPubMedView ArticleGoogle Scholar
- Lindenberg M, Solmaz G, Puttur F, Sparwasser T (2014) Mouse cytomegalovirus infection overrules T regulatory cell suppression on natural killer cells. Virol J 11:145PubMed CentralPubMedView ArticleGoogle Scholar
- Myers L, Joedicke JJ, Carmody AB, Messer RJ, Kassiotis G, Dudley JP et al (2013) IL-2-independent and TNF-alpha-dependent expansion of Vbeta5+ natural regulatory T cells during retrovirus infection. J Immunol 190:5485–5495PubMed CentralPubMedView ArticleGoogle Scholar
- Chesebro B, Bloom M, Wehrly K, Nishio J (1979) Persistence of infectious Friend virus in spleens of mice after spontaneous recovery from virus-induced erythroleukemia. J Virol 32:832–837PubMed CentralPubMedGoogle Scholar
- Zelinskyy G, Kraft AR, Schimmer S, Arndt T, Dittmer U (2006) Kinetics of CD8+ effector T cell responses and induced CD4+ regulatory T cell responses during Friend retrovirus infection. Eur J Immunol 36:2658–2670PubMedView ArticleGoogle Scholar
- Dietze KK, Zelinskyy G, Liu J, Kretzmer F, Schimmer S, Dittmer U (2013) Combining regulatory T cell depletion and inhibitory receptor blockade improves reactivation of exhausted virus-specific CD8+ T cells and efficiently reduces chronic retroviral loads. PLoS Pathog 9:e1003798PubMed CentralPubMedView ArticleGoogle Scholar
- Dietze KK, Zelinskyy G, Gibbert K, Schimmer S, Francois S, Myers L et al (2011) Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points. Proc Natl Acad Sci USA 108:2420–2425PubMed CentralPubMedView ArticleGoogle Scholar
- Zelinskyy G, Myers L, Dietze KK, Gibbert K, Roggendorf M, Liu J et al (2011) Virus-specific CD8+ T cells upregulate programmed death-1 expression during acute friend retrovirus infection but are highly cytotoxic and control virus replication. J Immunol 187:3730–3737PubMed CentralPubMedView ArticleGoogle Scholar
- Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G et al (2007) Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 204:57–63PubMed CentralPubMedView ArticleGoogle Scholar
- Huntington ND, Tabarias H, Fairfax K, Brady J, Hayakawa Y, Degli-Esposti MA et al (2007) NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation. J Immunol 178:4764–4770PubMedView ArticleGoogle Scholar
- Chiossone L, Chaix J, Fuseri N, Roth C, Vivier E, Walzer T (2009) Maturation of mouse NK cells is a 4-stage developmental program. Blood 113:5488–5496PubMedView ArticleGoogle Scholar
- Hayakawa Y, Smyth MJ (2006) CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity. J Immunol 176:1517–1524PubMedView ArticleGoogle Scholar
- Ghiringhelli F, Menard C, Terme M, Flament C, Taieb J, Chaput N et al (2005) CD4+ CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner. J Exp Med 202:1075–1085PubMed CentralPubMedView ArticleGoogle Scholar
- Smyth MJ, Teng MW, Swann J, Kyparissoudis K, Godfrey DI, Hayakawa Y (2006) CD4+ CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer. J Immunol 176:1582–1587PubMedView ArticleGoogle Scholar
- Manzke N, Akhmetzyanova I, Hasenkrug KJ, Trilling M, Zelinskyy G, Dittmer U (2013) CD4+ T cells develop antiretroviral cytotoxic activity in the absence of regulatory T cells and CD8+ T cells. J Virol 87:6306–6313PubMed CentralPubMedView ArticleGoogle Scholar
- Boyman O, Sprent J (2012) The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 12:180–190PubMedGoogle Scholar
- Boyman O, Kovar M, Rubinstein MP, Surh CD, Sprent J (2006) Selective stimulation of T cell subsets with antibody–cytokine immune complexes. Science 311:1924–1927PubMedView ArticleGoogle Scholar
- Belkaid Y, Rouse BT (2005) Natural regulatory T cells in infectious disease. Nat Immunol 6:353–360PubMedView ArticleGoogle Scholar
- Duppach J, Francois S, Joedicke JJ, Dittmer U, Kraft AR (2014) Expanded regulatory T cells in chronically Friend retrovirus infected mice suppress immunity to a mCMV superinfection. J Virol 88(23):13892–13896PubMed CentralPubMedView ArticleGoogle Scholar
- Lafaille FG, Pessach IM, Zhang SY, Ciancanelli MJ, Herman M, Abhyankar A et al (2012) Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells. Nature 491:769–773PubMed CentralPubMedGoogle Scholar
- Cao X, Cai SF, Fehniger TA, Song J, Collins LI, Piwnica-Worms DR et al (2007) Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity 27:635–646PubMedView ArticleGoogle Scholar
- Joedicke JJ, Dietze KK, Zelinskyy G, Dittmer U (2014) The phenotype and activation status of regulatory T cells during Friend retrovirus infection. Virologica Sinica 29:48–60PubMedView ArticleGoogle Scholar
- Sitrin J, Ring A, Garcia KC, Benoist C, Mathis D (2013) Regulatory T cells control NK cells in an insulitic lesion by depriving them of IL-2. J Exp Med 210:1153–1165PubMed CentralPubMedView ArticleGoogle Scholar
- Gasteiger G, Hemmers S, Firth MA, Le Floc’h A, Huse M, Sun JC et al (2013) IL-2-dependent tuning of NK cell sensitivity for target cells is controlled by regulatory T cells. J Exp Med 210:1167–1178PubMed CentralPubMedView ArticleGoogle Scholar
- Lee S, Margolin K (2011) Cytokines in cancer immunotherapy. Cancers 3:3856–3893PubMed CentralPubMedView ArticleGoogle Scholar
- Shevach EM (2012) Application of IL-2 therapy to target T regulatory cell function. Trends Immunol 33:626–632PubMed CentralPubMedView ArticleGoogle Scholar
- Newman RG, Dee MJ, Malek TR, Podack ER, Levy RB (2014) Heat shock protein vaccination and directed IL-2 therapy amplify tumor immunity rapidly following bone marrow transplantation in mice. Blood 123:3045–3055PubMed CentralPubMedView ArticleGoogle Scholar
- Kamimura D, Sawa Y, Sato M, Agung E, Hirano T, Murakami M (2006) IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb. J Immunol 177:306–314PubMedView ArticleGoogle Scholar
- Hamilton SE, Schenkel JM, Akue AD, Jameson SC (2010) IL-2 complex treatment can protect naive mice from bacterial and viral infection. J Immunol 185:6584–6590PubMed CentralPubMedView ArticleGoogle Scholar
- Levin AM, Bates DL, Ring AM, Krieg C, Lin JT, Su L et al (2012) Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484:529–533PubMed CentralPubMedView ArticleGoogle Scholar
- Margolin K, Atkins MB, Dutcher JP, Ernstoff MS, Smith JW 2nd, Clark JI et al (2007) Phase I trial of BAY 50-4798, an interleukin-2-specific agonist in advanced melanoma and renal cancer. Clin Cancer Res Off J Am Assoc Cancer Res 13:3312–3319View ArticleGoogle Scholar
- Dittmer U, Brooks DM, Hasenkrug KJ (1998) Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms. J Virol 72:6554–6558PubMed CentralPubMedGoogle Scholar