Distinct roles of NK cells in viral immunity during different phases of acute Friend retrovirus infection
© Littwitz et al.; licensee BioMed Central Ltd. 2013
Received: 13 May 2013
Accepted: 29 October 2013
Published: 1 November 2013
In many virus infections natural killer (NK) cells are critical for the rapid containment of virus replication. Polymorphisms in NK cell receptors as well as viral escape from NK cell responses are associated with pathogenesis and viral loads in HIV-infected individuals, emphasizing their importance in retroviral immunity. In contrast, NK cells of LCMV-infected mice dampened virus-specific T cell responses resulting in impaired virus control. Thus, the exact role of NK cells during different phases of viral infections remains elusive. In this study we characterized the NK cell response at different time points of an acute retroviral infection by using the Friend retrovirus (FV) mouse model.
Depletion of NK1.1+ cells during the initial phase of FV infection (3 to 4 days post infection) resulted in increased viral loads, which correlated with enhanced target cell killing and elevated NK cell effector functions. At days 7 to 15 post infection, NK and NKT cells did not contribute to anti-retroviral immunity. In the transition phase between acute and chronic infection (30 days post infection), NK and NKT cells exhibited an inhibitory role and their depletion resulted in reduced viral loads and significantly improved FV-specific CD8+ T cell responses.
Our results demonstrate an opposed activity of NK cells during retroviral infection. They were protective in the initial phase of infection, when adaptive T cell responses were not yet detectable, but were dispensable for viral immunity after T cell expansion. At later time points they exhibited regulatory functions in inhibiting virus-specific CD8+ T cell responses.
KeywordsNK cells Friend retrovirus Effector T cells Regulatory function Viral control
NK cells are cytotoxic cells of the innate immune system, which contribute to the control of many virus infections. They express a variety of different receptors on their surface driving their functions towards activating or inhibitory responses. NK cells mediate their effector functions through recognition and elimination of virus-infected cells. However, for several viral infections it was shown that NK cells suppress the adaptive immunity by killing virus–specific T cells [1–4]. This resulted in increased viral loads and might contribute to impaired viral clearance. On the other hand it has been demonstrated that HIV uses different mechanisms to escape NK cell responses. HIV modulates the ligand expression of the activating NK cell receptor NKG2D on infected cells resulting in reduced NK cell activation . Additionally, mutational adaptation of HIV has been shown to result in stimulation of inhibitory receptors on NK cells and subsequent escape from NK cell-mediated immune pressure . These opposed mechanisms suggest that NK cells may have distinct effects in different phases of viral infections. Here we analyzed these effects in the murine Friend retrovirus (FV) model by depletion of NK cells at different time points after retroviral infection. The antiviral role of NK cells during an acute FV infection has been investigated in several previous studies. However, almost all of these studies were performed with FV stocks containing Lactate dehydrogenase elevating virus (LDV). Only one recent paper with LDV-free FV showed that NK cells recognize infected target cells by interaction of RAE-1 and NKG2D . LDV induces a massive type I interferon response during acute infection, which we have shown to significantly modulate NK cell responses . Since LDV-free FV induces only barely detectable levels of type I interferons, the previous studies provide very little useful information on the role of NK cells in the immune control of FV infection. We therefore addressed this question in mice that were infected with a LDV-free FV stock.
Kinetics of FV infection in different lymphoid organs
To analyze the impact of NK cells on FV replication, we depleted NK cells during different phases of infection. Here, we focused on three different time points of acute FV infection. 1) The initial phase (3–4 dpi), when only NK cells are able to kill virus-infected cells because T cell responses are not yet developed . 2) At the peak of virus replication (7–15 dpi), when virus-specific T cells are detectable and eliminate infected target cells . 3) The late phase (30 dpi), when viral loads are reduced but FV-induced leukemia starts to develop. Then cytotoxic CD8+ T cells become dysfunctional in FV infection .
Anti-retroviral effects of NK cells during initial FV infection
No effect of NK cells during the peak FV replication
Next, we examined the function of NK cells 7–15 dpi, when T cells expand and control FV replication [9, 10]. Depletion of NK and NKT cells during this phase of infection did not influence viral loads in both investigated organs, which was shown by an IC assay (Figure 2A-2B) and RT-PCR analysis (data not shown). However, at that time point both cell subsets started to express high levels of CD69 (Figure 3A, 3B and data not shown). The expression of the functional molecules granzyme B, interferon-γ (IFN), or TRAIL were elevated only in NK cells from spleen or bone marrow (Figure 3C-3E), but isolated NK cells failed to eliminate target cells (Figure 3F and 3G) which correlated with the findings from the depletion experiments (Figure 2A-2B). Thus at 7 to 15 dpi NK cells and NKT cells did not contribute to antiviral immunity, which is mainly mediated by CD8+ T cells during peak viral replication .
Negative effects of NK cells during the late phase of acute FV infection
During the late phase of FV infection (30 dpi) susceptible mice start to develop a lethal erythroleukemia. Then virus-infected as well as transformed cells are potential targets for NK cells. To evaluate their role during this phase, we performed depletion experiments starting at 20 dpi. Surprisingly, application of α-NK1.1 antibody resulted in a significant reduction of viral loads at 30 dpi, which was observed by an IC assay (Figure 2A-2B) and RT-PCR analysis (data not shown). An 8-fold decrease of the viral loads in the bone marrow (Figure 2A) and a 4-fold reduction in the spleen (Figure 2B) was observed compared to control mice. NK cells were still activated (CD69+) at this late time point (Figure 3A and 3B) and expressed elevated levels of granzyme B (Figure 3C) and IFN-γ (Figure 3D). TRAIL was not detected on NK cells from mice infected for 30 days (Figure 3E). Next, we analyzed if up-regulation of effector molecules correlated with the cytotoxic potential of NK cells during late acute FV infection. Figure 3F and 3G show that NK cells isolated 30 dpi significantly lysed target cells. 30 dpi NK cells were activated and had an effector phenotype, which allowed them to specifically kill target cells in vitro. Surprisingly, NK and NKT cells did not contribute to viral immunity but rather negatively influenced viral loads during the late phase of acute FV infection.
NK cell depletion improved specific CD8+T cell responses in FV-infected mice
In this report, we analyzed NK cell effector functions and their impact on viral replication and adaptive immune responses during acute retroviral infection. Altogether, we identified an antiviral role of NK cells or NKT cells during the initial FV infection (3–4 dpi), which might contribute to the early control of viral replication. Others could already show that mice deficient in NKT cells were more susceptible in controlling some viruses like HSV-1/2, MCMV, and EMCV (reviewed in ). Thus, either NK cells or NKT cells might participate in the early antiviral immunity against FV. Later during infection, CD8+ T cells become the key players of anti-retroviral immunity  and replace NK and NKT cell responses. However, the strong CD8+ T cell responses might have to be counter-regulated in some virus infections to prevent immunopathology. There is growing evidence that NK cells and not NKT cells play a role in this regulation. In the LCMV-model, it was shown that depletion of NK cells alone enhanced T cell responses and thus mice could control viral replication and thereby prevent chronic infection . The NK cell-mediated inhibition of CD8+ T cells in this model depended on NKG2D binding and perforin production. Others reported that activated NK cells eliminate CD4+ T cells in a perforin-dependent manner, which might indirectly affect CD8+ T cell function and exhaustion during LCMV infection . We did not observe an effect of NK cell depletion on CD4+ T cell responses suggesting a direct suppressive effect of NK cells on CD8+ T cells in our model. Others demonstrated that NK cell depletion during high-dose influenza infection improved the survival rate of infected mice which was not seen in medium- or low-dose influenza infections [3, 13, 14]. The data indicate that viral loads might predict the effector functions of NK cells. Moreover, during herpes virus infection, a regulatory role of NK cells was described. It was shown that a strong NK cell response can limit T cell responses in MCMV infection [15, 16]. Altogether, it seems that the balance of NK and T cell responses seems to be critical for viral immunity and the impact of NK cells depends on the type and dose of virus as well as the phase of virus infection. Thus, it is very important to further investigate the factors that influence NK cell regulatory functions.
This work was supported by a grant from the University Hospital Essen (IFORES).
- Waggoner SN, Cornberg M, Selin LK, Welsh RM: Natural killer cells act as rheostats modulating antiviral T cells. Nature. 2012, 481 (7381): 394-398.Google Scholar
- Lang PA, Lang KS, Xu HC, Grusdat M, Parish IA, Recher M, Elford AR, Dhanji S, Shaabani N, Tran CW, et al: Natural killer cell activation enhances immune pathology and promotes chronic infection by limiting CD8+ T cellimmunity. Proc Natl Acad Sci USA. 2012, 109 (4): 1210-1215. 10.1073/pnas.1118834109.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou G, Juang SW, Kane KP: NK cells exacerbate the pathology of influenza virus infection in mice. Eur J Iimmunol. 2013, 43 (4): 929-938. 10.1002/eji.201242620.View ArticleGoogle Scholar
- Soderquest K, Walzer T, Zafirova B, Klavinskis LS, Polic B, Vivier E, Lord GM, Martin-Fontecha A: Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses. J Iimmunol. 2011, 186 (6): 3304-3308. 10.4049/jimmunol.1004122.View ArticleGoogle Scholar
- Matusali G, Tchidjou HK, Pontrelli G, Bernardi S, D’Ettorre G, Vullo V, Buonomini AR, Andreoni M, Santoni A, Cerboni C, et al: Soluble ligands for the NKG2D receptor are released during HIV-1 infection and impair NKG2D expression and cytotoxicity of NK cells. FASEB J. 2013, 27 (6): 2440-2450. 10.1096/fj.12-223057.View ArticlePubMedGoogle Scholar
- Alter G, Heckerman D, Schneidewind A, Fadda L, Kadie CM, Carlson JM, Oniangue-Ndza C, Martin M, Li B, Khakoo SI, et al: HIV-1 adaptation to NK-cell-mediated immune pressure. Nature. 2011, 476 (7358): 96-100. 10.1038/nature10237.PubMed CentralView ArticlePubMedGoogle Scholar
- Ogawa T, Tsuji-Kawahara S, Yuasa T, Kinoshita S, Chikaishi T, Takamura S, Matsumura H, Seya T, Saga T, Miyazawa M: Natural killer cells recognize friend retrovirus-infected erythroid progenitor cells through NKG2D-RAE-1 interactions In Vivo. J Virol. 2011, 85 (11): 5423-5435. 10.1128/JVI.02146-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Gibbert K, Joedicke JJ, Meryk A, Trilling M, Francois S, Duppach J, Kraft A, Lang KS, Dittmer U: Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection. PLoS Pathog. 2012, 8 (8): e1002868-10.1371/journal.ppat.1002868.PubMed CentralView ArticlePubMedGoogle Scholar
- Zelinskyy G, Dietze KK, Husecken YP, Schimmer S, Nair S, Werner T, Gibbert K, Kershaw O, Gruber AD, Sparwasser T, et al: 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. 2009, 114 (15): 3199-3207. 10.1182/blood-2009-03-208736.View ArticlePubMedGoogle Scholar
- Manzke N, Akhmetzyanova I, Hasenkrug KJ, Trilling M, Zelinskyy G, Dittmer U: CD4+ T cells develop antiretroviral cytotoxic activity in the absence of regulatory T cells and CD8+ T cells. J Virol. 2013, 87 (11): 6306-6313. 10.1128/JVI.00432-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Shimizu T, Uenishi H, Teramura Y, Iwashiro M, Kuribayashi K, Tamamura H, Fujii N, Yamagishi H: Fine structure of a virus-encoded helper T cellepitope expressed on FBL-3 tumor cells. J Virol. 1994, 68 (12): 7704-7708.PubMed CentralPubMedGoogle Scholar
- Diana J, Lehuen A: NKT cells: friend or foe during viral infections?. Eur J Immunol. 2009, 39 (12): 3283-3291. 10.1002/eji.200939800.View ArticlePubMedGoogle Scholar
- Gazit R, Gruda R, Elboim M, Arnon TI, Katz G, Achdout H, Hanna J, Qimron U, Landau G, Greenbaum E, et al: Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1. Nat Immunol. 2006, 7 (5): 517-523. 10.1038/ni1322.View ArticlePubMedGoogle Scholar
- Stein-Streilein J, Guffee J: In vivo treatment of mice and hamsters with antibodies to asialo GM1 increases morbidity and mortality to pulmonary influenza infection. J Immunol. 1986, 136 (4): 1435-1441.PubMedGoogle Scholar
- Andrews DM, Estcourt MJ, Andoniou CE, Wikstrom ME, Khong A, Voigt V, Fleming P, Tabarias H, Hill GR, van der Most RG, et al: Innate immunity defines the capacity of antiviral T cells to limit persistent infection. J Exp Med. 2010, 207 (6): 1333-1343. 10.1084/jem.20091193.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee SH, Kim KS, Fodil-Cornu N, Vidal SM, Biron CA: Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection. J Exp Med. 2009, 206 (10): 2235-2251. 10.1084/jem.20082387.PubMed CentralView ArticlePubMedGoogle Scholar
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