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ScienceDirect Animal models of Epstein Barr virus infection Cornelia Gujer, Bithi Chatterjee, Vanessa Landtwing, Ana Raykova, Donal McHugh and Christian Mu¨nz Epstein Barr virus (EBV) was the first human tumor virus to be described. Despite its discovery now more than fifty years ago, immune control of this virus is still not very well understood and no vaccine is available. This knowledge gap is due in part to the lack of a preclinical small animal model which can faithfully recapitulate EBV infection and immune control, and would allow testing of EBV specific vaccine candidates. With the advent of mice with reconstituted human immune system compartments (HIS mice) during the past decade this is changing. We will discuss which aspects of EBV infection and its immune control can already be modeled in HIS mice, and which shortcomings still need to be overcome in order to recapitulate the immunobiology of oncogenic EBV infection. Address Viral Immunobiology, Institute for Experimental Immunology, University of Zu¨rich, Switzerland Corresponding author: Mu¨nz, Christian ([email protected])
Current Opinion in Virology 2015, 13:6–10 This review comes from a themed issue on Animal models for viral diseases Edited by Christopher Walker and Alex Ploss
http://dx.doi.org/10.1016/j.coviro.2015.03.014 1879-6257/# 2015 Elsevier B.V. All rights reserved.
Introduction Epstein Barr virus (EBV) is a highly successful g-herpesvirus in the human population. It persistently infects more than 90% of human adults, and is controlled by cellmediated immune responses in most infected individuals for life [1]. Primary infection is thought to occur via saliva exchange and transepithelial access of viral particles to B cells in submucosal secondary lymphoid tissues. In these target cells, EBV initially replicates vertically through proliferation of the latently infected B cells. Expression of six EBV nuclear antigens (EBNAs), two latent membrane proteins (LMPs), two EBV encoded RNAs (EBERs) and around 40 microRNAs drives B cells into proliferation [2]. These activated B cells then undergo differentiation leading to EBV persistence in long-lived memory B cells and reactivation into lytic replication and virus production in plasma cells [3]. The epigenetic silencing of the EBV Current Opinion in Virology 2015, 13:6–10
genome and the down-regulation of latent EBV antigen expression, which is associated with B cell differentiation, are both required for the function of Zta (BZLF1), which initiates transcription of around 80 lytic EBV genes [4]. Reactivation most likely occurs through B cell receptor triggering by the cognate antigen at submucosal sites. There, EBV can infect epithelial cells via the basolateral side for an additional round of lytic replication and subsequent shedding into the saliva for transmission [5,6]. Because of possibly only vertical replication via B cell proliferation initially, EBV viral loads only peak and can cause symptoms after four to six weeks of primary infection [7,8]. This symptomatic primary EBV infection, which preferentially occurs when primary EBV infection is delayed into adolescence, is called infectious mononucleosis (IM) or Pfeiffer’s glandular fever [9]. The associated symptoms are linked to the immunopathologic expansion of cytotoxic CD8+ T cells, which are also thought to primarily exert immune control of the virus [1]. Consistent with this important role of CD8+ T cells, immunosuppression that compromises T cell function, leading to uncontrolled EBV infected B cell proliferation, can be treated by adoptively transferring EBV specific T cell lines; individuals that lack EBV specific antibodies can still control persistent EBV infection [10,11]. Loss of this EBV specific immune control contributes to the development of EBV associated malignancies. Indeed, this virus was discovered more than fifty years ago as the first human tumorvirus [12,13]. It is now known to be associated with B cell derived tumors like Burkitt’s and Hodgkin’s lymphomas, as well as epithelial cell derived tumors like nasopharyngeal and a subset of gastric carcinomas [2]. Apart from T cell mediated immune control, however, little is known about immune compartments that are additionally required to establish and maintain EBV specific immune control. At the time of clinical presentation of IM, the innate immune responses that precede the associated massive CD8+ T cell expansion have left only few traces. Moreover, the exclusive tropism of EBV for humans made it impossible to study this important human pathogen in a preclinical in vivo model system until recently. In this review, we will summarize and outline to which extent mice with human immune system components, that are reconstituted from neonatally transferred human CD34+ hematopoietic progenitor cells, (HIS mice) [14] have and can be used to address these questions.
EBV infection and tumorigenesis in HIS mice EBV was the first human pathogen to be studied in HIS mice [15]. Depending on the dose, infection of HIS mice www.sciencedirect.com
EBV infection in HIS mice Gujer et al.
with EBV can range from asymptomatic viral persistence in B cells to lymphoproliferative disease (LPD), similar to what is seen in immunocompromised individuals [16,17]. Mice inoculated with an intermediate dose of the most commonly used laboratory EBV strain, B95-8, isolated from an American IM patient [18], results in viremia after four to six weeks (Figure 1), progressive weight loss and splenomegaly. This is due to EBV-infected B cell proliferation and reactive CD8+ T cell expansion (Figure 1) [19]. As such, acute, symptomatic EBV infection seen in IM patients can be modeled in HIS mice. Latent and lytic EBV infection may occur to different extents in HIS mice, depending on the viral stain used for inoculation. Upon infection with the B95-8 strain, only a minority of infected B cells enter the lytic replication cycle, as evidenced by the low levels of lytic immediate early transactivator BZLF1 expression [20–22] and the similar to wild-type viremia profile during infection with a BZLF1 KO virus (Figure 1) [20]. The majority of infected B cells express all eight latent EBV proteins. This has been confirmed by immunohistochemical co-staining for EBNA2 and LMP1 and an abundance of Cp promotor driven EBNA1 mRNA expression [16,23]. This pattern of EBV gene expression is termed latency III and is commonly found in polymorphic post-transplant lymphoproliferative disease or AIDS-associated Diffuse Large B Cell Lymphoma of the immunoblastic type [2]. It has been suggested that B cells expressing latencies I and II with reduced latent EBV antigen expression (typically found in Burkitt lymphoma and Hodgkin lymphoma, respectively) are also present upon HIS mouse infection Figure 1
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with EBV [22–24]. However, these seem to contribute to a small percentage of all EBV-infected B cells in conventional HIS mice. The use of mice in which both human fetal CD34+ hematopoietic progenitor cells and thymus/ liver tissue are transplanted (BLT mice) may be more likely to support the development of B cells harboring latencies I and II, possibly due to better immune restriction of latency III B cells by T cells and better secondary lymphoid tissue development in BLT mice of the NODscid mouse background [22,25]. Without germinal center development in secondary lymphoid tissues HIS mice primarily harbor transitional and naı¨ve B cells [26]. Furthermore, the HIS mouse model has proved useful to study the in vivo biology of specific latent and lytic EBV gene products by employing recombinant knock-out viruses [20,22,27,28] or to compare different EBV strains [21]. Along these lines, infection of HIS mice with an EBV deficient in EBNA3B, shown to be dispensable for transformation in vitro, resulted in increased LPD and tumor formation due to aggressive B cell proliferation and reduced secretion of CXCL10, a T cell attracting chemokine, thus identifying EBNA3B as a viral tumor suppressor [27]. In another study HIS mice were infected with an EBV strain called M81, isolated from a Chinese nasopharyngeal carcinoma patient, and this resulted in an increased frequency of lytically replicating cells compared to B95-8 and higher viremia [21]. Lytic activity is, however, not required for stable infection of HIS mice [22], but surprisingly may enhance the efficiency of tumorigenesis [20,22]. Finally, complete latency III transformation and the expression of LMP1 does also not seem to be required for EBV persistence, because T cell help might substitute for LMP1 function [28]. Thus, lytic and latency III infection by EBV and their respective associated malignancies can be modeled in HIS mice.
BZLF1+ BZLF1– EBV titer
Innate immune control of EBV infection in HIS mice
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Current Opinion in Virology
Dynamics of wild-type and lytic replication deficient EBV infection and immune control in HIS mice. Viremia of wild-type (BZLF1+) and lytic replication deficient (BZLF1 ) B95-8 EBV infection differ only transiently at early time points after EBV infection (mainly week three). NK cell expansion peaks at week four controlling lytic EBV replication, while EBV infection is prevented from further increasing by lytic and latent EBV antigen specific T cell responses at weeks five and six after inoculation of HIS mice. www.sciencedirect.com
Since innate responses to EBV might be responsible for preventing symptoms of IM, it is important to increase the understanding of these early responses to EBV. Innate immune compartments such as dendritic cells (DCs) and natural killer (NK) cells were suggested to play a role in the control of EBV infection. Conventional DCs (cDCs) and the plasmacytoid DCs (pDCs) as well as NK cells reconstitute in HIS mouse models [29–33]. These DC subsets have been shown to sense EBV using different PRRs. While pDCs recognize unmethylated CpG DNA motifs present in the dsDNA of EBV particles via Toll-like receptor (TLR) 9 [34,35], cDCs can recognize EBERs that are released from infected cells using TLR3 or the intracellular receptor RIG-I [36]. PDCs were required for immune control of EBV infection in a transfer model of human peripheral blood mononuclear cells (PBMCs) into immunocompromised mice. Current Opinion in Virology 2015, 13:6–10
8 Animal models for viral diseases
Mice that received PBMCs that were depleted of pDCs showed less control of EBV infection compared with mice that received pDC enriched PBMCs, as reflected by delayed mortality. PDCs were shown to be involved in the activation of T cells via cell to cell contact and mediated via TLR9 [37]. In addition, pDCs were shown to secrete interferon (IFN) a in response to EBV. IFNa was shown in vitro to restrict EBV transformation early after infection [38]. Human monocyte-derived DCs are able to prime protective EBV specific T cell responses in vitro [39], however, the role of the other DC subsets in EBV infection has not been studied yet in vivo. In addition to their antigen-presenting role in the activation of T cells, DCs have the ability to activate other innate immune cells including NK cells by cell to cell contact and the production of cytokines [40–42]. In HIS mice the NK cell compartment contains NKp46+ CD56 cells that upregulate CD56 upon IL-15 stimulation in vitro and are usually only found in cord blood. The functional competence of the more mature CD56dim NK cell compartment is decreased compared to PBMC-derived NK cells and can be rescued by preactivation with IL-15 in vitro and poly(I:C) in vivo [29]. In humans, NK cells have been suggested to determine susceptibility to EBV infection [43,44]. Accordingly, HIS mice depleted of NK cells before infection presented with higher viral titers. This disease phenotype of NK cell depleted animals was rescued when infecting mice with an EBV strain deficient for lytic replication. Additionally, the increased levels of lytic replication and lack of NK cells lead to a massive CD8+ T cell expansion and splenomegaly. Overall NK cell depleted animals had exacerbated disease progression, including higher weight loss and tumorigenesis. This data supports the idea that NK cells in the early stages of differentiation are crucial for the innate immune control of lytic EBV replication, and their decreased frequency after the first decade of human life might predispose for IM [19,45].
Adaptive immune control of EBV infection in HIS mice With recent advances, the study of adaptive immune control of EBV infection in HIS mice has yielded interesting results. Both CD4+ and CD8+ T cell compartments reconstitute in HIS mice with varying degrees of success depending on the model used [15,16]. In NOD-scid gc / mice with an HLA-A*0201 transgene, CD8+ T cells have been found with similar latent and lytic EBV specificities as have been described in humans [16,46]. In the absence of this transgene, subdominant epitopes, as characterized in humans, seem to predominate. CD8+ T cell numbers expand during intermediate dose EBV infection of HIS mice, similar to patients suffering from IM. Reconstituted T cells in HIS animals exert a significant degree of immune control during EBV infection. T cells that arise in response to EBV infection produce IFNg in response Current Opinion in Virology 2015, 13:6–10
to both cognate antigens [16] and EBV transformed B cells (LCLs) [15,17,31], and specific CD4+ and CD8+ clones primed in EBV infected animals are able to lyse LCLs [16,30]. Indeed, LCL recognition can be reduced upon the addition of MHC Class I/II blocking antibodies [16,17,31]. The introduction of lytic BMLF1 antigen specific CD8+ T cell clones appears to transiently control EBV viremia in HIS mice just before NK cell expansion and may reduce the incidence of lytic antigen expressing B cells in the spleens of EBV infected animals [20]. This was not observed upon the introduction of latent LMP2 antigen specific CD8+ T cells, indicating a protective function of lytic antigen specific T cells [20]. Studies in HIS mice have also demonstrated that CD8+ T cells work in concert with NK cells during acute EBV infection (Figure 1) [19]. CD8+ T cell expansion is reduced by the presence of NK cells and the depletion of NK cells leads to elevated CD8+ and CD4+ T cell frequencies. Interestingly, the depletion of both CD8+ T cells and NK cells leads to elevated EBV viral loads compared to depleting NK cells alone [19]. T cells also play an important role in controlling EBV tumorigenesis [16,47]. The depletion of CD8+ T cells results in EBV mediated tumorigenesis in HIS mice and elevated viral loads and the depletion of both CD4+ and CD8+ subsets resulted in further elevated viral loads and increased tumorigenesis [16]. While the interrogation of T cell responses during EBV infection is possible in HIS animals, the examination of B cell responses is still difficult. This is likely due to poor germinal center organization [30] and incomplete B cell maturation in HIS animals. Weak IgM and IgG responses have been observed in HIS animals following reconstitution [15,48,49], and EBV specific responses are largely lacking, with the exception of IgM production upon EBV infection [17]. Efforts to promote better germinal center organization and immunoglobulin class switching may generate superior B cell responses [26], and together with the use of MHC Class II transgenic animals may ensure more effective B cell/T cell cross talk.
Conclusions HIS mice are able to model certain important aspects of EBV infection and immune control. These include latency III and low levels of lytic EBV infection, LPD and cellmediated immune control by NK and T cells. Their main limitations are weak to absent humoral immune responses and a lack of routine genetic manipulations of the reconstituted human immune system compartments thus far. Furthermore, the compromised secondary lymphoid organ development in most currently used HIS mouse models, impedes not only isotype switched antibody responses, but also programs of EBV infection that might be dependent on germinal center reactions (latencies I and II) and also lymphocyte differentiation, like immature NK cell compartments that are enriched in these www.sciencedirect.com
EBV infection in HIS mice Gujer et al.
anatomical locations. However, if these challenges can be overcome, both immune responses and their protective function during infection with human specific pathogens like EBV can be efficiently studied and vaccine candidates can be explored for their capacity to induce these protective responses.
15. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A, Manz MG: Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 2004, 304:104-107.
Acknowledgements
17. Yajima M, Imadome K, Nakagawa A, Watanabe S, Terashima K, Nakamura H, Ito M, Shimizu N, Honda M, Yamamoto N, Fujiwara S: A new humanized mouse model of Epstein-Barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses. J Infect Dis 2008, 198:673-682.
CG is supported by the Marie Heim-Vo¨gtlin program of the Swiss National Science foundation (SNF; PMPDP3_145504) and DM by a fellowship awarded by the Swiss Academy of Medical Sciences and the SNF (323530_145247). Our laboratory is financed by grants to CM from Cancer Research Switzerland (KFS-3234-08-2013), Worldwide Cancer Research (14-1033), KFSPMS and KFSPHHLD of the University of Zurich, the Baugarten Foundation, the Sobek Foundation, Fondation Acteria, the Swiss Vaccine Research Institute and the SNF (310030_143979 and CRSII3_136241).
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ScienceDirect Animal models of Epstein Barr virus infection Cornelia Gujer, Bithi Chatterjee, Vanessa Landtwing, Ana Raykova, Donal McHugh and Christian Mu¨nz Epstein Barr virus (EBV) was the first human tumor virus to be described. Despite its discovery now more than fifty years ago, immune control of this virus is still not very well understood and no vaccine is available. This knowledge gap is due in part to the lack of a preclinical small animal model which can faithfully recapitulate EBV infection and immune control, and would allow testing of EBV specific vaccine candidates. With the advent of mice with reconstituted human immune system compartments (HIS mice) during the past decade this is changing. We will discuss which aspects of EBV infection and its immune control can already be modeled in HIS mice, and which shortcomings still need to be overcome in order to recapitulate the immunobiology of oncogenic EBV infection. Address Viral Immunobiology, Institute for Experimental Immunology, University of Zu¨rich, Switzerland Corresponding author: Mu¨nz, Christian ([email protected])
Current Opinion in Virology 2015, 13:6–10 This review comes from a themed issue on Animal models for viral diseases Edited by Christopher Walker and Alex Ploss
http://dx.doi.org/10.1016/j.coviro.2015.03.014 1879-6257/# 2015 Elsevier B.V. All rights reserved.
Introduction Epstein Barr virus (EBV) is a highly successful g-herpesvirus in the human population. It persistently infects more than 90% of human adults, and is controlled by cellmediated immune responses in most infected individuals for life [1]. Primary infection is thought to occur via saliva exchange and transepithelial access of viral particles to B cells in submucosal secondary lymphoid tissues. In these target cells, EBV initially replicates vertically through proliferation of the latently infected B cells. Expression of six EBV nuclear antigens (EBNAs), two latent membrane proteins (LMPs), two EBV encoded RNAs (EBERs) and around 40 microRNAs drives B cells into proliferation [2]. These activated B cells then undergo differentiation leading to EBV persistence in long-lived memory B cells and reactivation into lytic replication and virus production in plasma cells [3]. The epigenetic silencing of the EBV Current Opinion in Virology 2015, 13:6–10
genome and the down-regulation of latent EBV antigen expression, which is associated with B cell differentiation, are both required for the function of Zta (BZLF1), which initiates transcription of around 80 lytic EBV genes [4]. Reactivation most likely occurs through B cell receptor triggering by the cognate antigen at submucosal sites. There, EBV can infect epithelial cells via the basolateral side for an additional round of lytic replication and subsequent shedding into the saliva for transmission [5,6]. Because of possibly only vertical replication via B cell proliferation initially, EBV viral loads only peak and can cause symptoms after four to six weeks of primary infection [7,8]. This symptomatic primary EBV infection, which preferentially occurs when primary EBV infection is delayed into adolescence, is called infectious mononucleosis (IM) or Pfeiffer’s glandular fever [9]. The associated symptoms are linked to the immunopathologic expansion of cytotoxic CD8+ T cells, which are also thought to primarily exert immune control of the virus [1]. Consistent with this important role of CD8+ T cells, immunosuppression that compromises T cell function, leading to uncontrolled EBV infected B cell proliferation, can be treated by adoptively transferring EBV specific T cell lines; individuals that lack EBV specific antibodies can still control persistent EBV infection [10,11]. Loss of this EBV specific immune control contributes to the development of EBV associated malignancies. Indeed, this virus was discovered more than fifty years ago as the first human tumorvirus [12,13]. It is now known to be associated with B cell derived tumors like Burkitt’s and Hodgkin’s lymphomas, as well as epithelial cell derived tumors like nasopharyngeal and a subset of gastric carcinomas [2]. Apart from T cell mediated immune control, however, little is known about immune compartments that are additionally required to establish and maintain EBV specific immune control. At the time of clinical presentation of IM, the innate immune responses that precede the associated massive CD8+ T cell expansion have left only few traces. Moreover, the exclusive tropism of EBV for humans made it impossible to study this important human pathogen in a preclinical in vivo model system until recently. In this review, we will summarize and outline to which extent mice with human immune system components, that are reconstituted from neonatally transferred human CD34+ hematopoietic progenitor cells, (HIS mice) [14] have and can be used to address these questions.
EBV infection and tumorigenesis in HIS mice EBV was the first human pathogen to be studied in HIS mice [15]. Depending on the dose, infection of HIS mice www.sciencedirect.com
EBV infection in HIS mice Gujer et al.
with EBV can range from asymptomatic viral persistence in B cells to lymphoproliferative disease (LPD), similar to what is seen in immunocompromised individuals [16,17]. Mice inoculated with an intermediate dose of the most commonly used laboratory EBV strain, B95-8, isolated from an American IM patient [18], results in viremia after four to six weeks (Figure 1), progressive weight loss and splenomegaly. This is due to EBV-infected B cell proliferation and reactive CD8+ T cell expansion (Figure 1) [19]. As such, acute, symptomatic EBV infection seen in IM patients can be modeled in HIS mice. Latent and lytic EBV infection may occur to different extents in HIS mice, depending on the viral stain used for inoculation. Upon infection with the B95-8 strain, only a minority of infected B cells enter the lytic replication cycle, as evidenced by the low levels of lytic immediate early transactivator BZLF1 expression [20–22] and the similar to wild-type viremia profile during infection with a BZLF1 KO virus (Figure 1) [20]. The majority of infected B cells express all eight latent EBV proteins. This has been confirmed by immunohistochemical co-staining for EBNA2 and LMP1 and an abundance of Cp promotor driven EBNA1 mRNA expression [16,23]. This pattern of EBV gene expression is termed latency III and is commonly found in polymorphic post-transplant lymphoproliferative disease or AIDS-associated Diffuse Large B Cell Lymphoma of the immunoblastic type [2]. It has been suggested that B cells expressing latencies I and II with reduced latent EBV antigen expression (typically found in Burkitt lymphoma and Hodgkin lymphoma, respectively) are also present upon HIS mouse infection Figure 1
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with EBV [22–24]. However, these seem to contribute to a small percentage of all EBV-infected B cells in conventional HIS mice. The use of mice in which both human fetal CD34+ hematopoietic progenitor cells and thymus/ liver tissue are transplanted (BLT mice) may be more likely to support the development of B cells harboring latencies I and II, possibly due to better immune restriction of latency III B cells by T cells and better secondary lymphoid tissue development in BLT mice of the NODscid mouse background [22,25]. Without germinal center development in secondary lymphoid tissues HIS mice primarily harbor transitional and naı¨ve B cells [26]. Furthermore, the HIS mouse model has proved useful to study the in vivo biology of specific latent and lytic EBV gene products by employing recombinant knock-out viruses [20,22,27,28] or to compare different EBV strains [21]. Along these lines, infection of HIS mice with an EBV deficient in EBNA3B, shown to be dispensable for transformation in vitro, resulted in increased LPD and tumor formation due to aggressive B cell proliferation and reduced secretion of CXCL10, a T cell attracting chemokine, thus identifying EBNA3B as a viral tumor suppressor [27]. In another study HIS mice were infected with an EBV strain called M81, isolated from a Chinese nasopharyngeal carcinoma patient, and this resulted in an increased frequency of lytically replicating cells compared to B95-8 and higher viremia [21]. Lytic activity is, however, not required for stable infection of HIS mice [22], but surprisingly may enhance the efficiency of tumorigenesis [20,22]. Finally, complete latency III transformation and the expression of LMP1 does also not seem to be required for EBV persistence, because T cell help might substitute for LMP1 function [28]. Thus, lytic and latency III infection by EBV and their respective associated malignancies can be modeled in HIS mice.
BZLF1+ BZLF1– EBV titer
Innate immune control of EBV infection in HIS mice
NK cells CD8+ T cells
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1
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Current Opinion in Virology
Dynamics of wild-type and lytic replication deficient EBV infection and immune control in HIS mice. Viremia of wild-type (BZLF1+) and lytic replication deficient (BZLF1 ) B95-8 EBV infection differ only transiently at early time points after EBV infection (mainly week three). NK cell expansion peaks at week four controlling lytic EBV replication, while EBV infection is prevented from further increasing by lytic and latent EBV antigen specific T cell responses at weeks five and six after inoculation of HIS mice. www.sciencedirect.com
Since innate responses to EBV might be responsible for preventing symptoms of IM, it is important to increase the understanding of these early responses to EBV. Innate immune compartments such as dendritic cells (DCs) and natural killer (NK) cells were suggested to play a role in the control of EBV infection. Conventional DCs (cDCs) and the plasmacytoid DCs (pDCs) as well as NK cells reconstitute in HIS mouse models [29–33]. These DC subsets have been shown to sense EBV using different PRRs. While pDCs recognize unmethylated CpG DNA motifs present in the dsDNA of EBV particles via Toll-like receptor (TLR) 9 [34,35], cDCs can recognize EBERs that are released from infected cells using TLR3 or the intracellular receptor RIG-I [36]. PDCs were required for immune control of EBV infection in a transfer model of human peripheral blood mononuclear cells (PBMCs) into immunocompromised mice. Current Opinion in Virology 2015, 13:6–10
8 Animal models for viral diseases
Mice that received PBMCs that were depleted of pDCs showed less control of EBV infection compared with mice that received pDC enriched PBMCs, as reflected by delayed mortality. PDCs were shown to be involved in the activation of T cells via cell to cell contact and mediated via TLR9 [37]. In addition, pDCs were shown to secrete interferon (IFN) a in response to EBV. IFNa was shown in vitro to restrict EBV transformation early after infection [38]. Human monocyte-derived DCs are able to prime protective EBV specific T cell responses in vitro [39], however, the role of the other DC subsets in EBV infection has not been studied yet in vivo. In addition to their antigen-presenting role in the activation of T cells, DCs have the ability to activate other innate immune cells including NK cells by cell to cell contact and the production of cytokines [40–42]. In HIS mice the NK cell compartment contains NKp46+ CD56 cells that upregulate CD56 upon IL-15 stimulation in vitro and are usually only found in cord blood. The functional competence of the more mature CD56dim NK cell compartment is decreased compared to PBMC-derived NK cells and can be rescued by preactivation with IL-15 in vitro and poly(I:C) in vivo [29]. In humans, NK cells have been suggested to determine susceptibility to EBV infection [43,44]. Accordingly, HIS mice depleted of NK cells before infection presented with higher viral titers. This disease phenotype of NK cell depleted animals was rescued when infecting mice with an EBV strain deficient for lytic replication. Additionally, the increased levels of lytic replication and lack of NK cells lead to a massive CD8+ T cell expansion and splenomegaly. Overall NK cell depleted animals had exacerbated disease progression, including higher weight loss and tumorigenesis. This data supports the idea that NK cells in the early stages of differentiation are crucial for the innate immune control of lytic EBV replication, and their decreased frequency after the first decade of human life might predispose for IM [19,45].
Adaptive immune control of EBV infection in HIS mice With recent advances, the study of adaptive immune control of EBV infection in HIS mice has yielded interesting results. Both CD4+ and CD8+ T cell compartments reconstitute in HIS mice with varying degrees of success depending on the model used [15,16]. In NOD-scid gc / mice with an HLA-A*0201 transgene, CD8+ T cells have been found with similar latent and lytic EBV specificities as have been described in humans [16,46]. In the absence of this transgene, subdominant epitopes, as characterized in humans, seem to predominate. CD8+ T cell numbers expand during intermediate dose EBV infection of HIS mice, similar to patients suffering from IM. Reconstituted T cells in HIS animals exert a significant degree of immune control during EBV infection. T cells that arise in response to EBV infection produce IFNg in response Current Opinion in Virology 2015, 13:6–10
to both cognate antigens [16] and EBV transformed B cells (LCLs) [15,17,31], and specific CD4+ and CD8+ clones primed in EBV infected animals are able to lyse LCLs [16,30]. Indeed, LCL recognition can be reduced upon the addition of MHC Class I/II blocking antibodies [16,17,31]. The introduction of lytic BMLF1 antigen specific CD8+ T cell clones appears to transiently control EBV viremia in HIS mice just before NK cell expansion and may reduce the incidence of lytic antigen expressing B cells in the spleens of EBV infected animals [20]. This was not observed upon the introduction of latent LMP2 antigen specific CD8+ T cells, indicating a protective function of lytic antigen specific T cells [20]. Studies in HIS mice have also demonstrated that CD8+ T cells work in concert with NK cells during acute EBV infection (Figure 1) [19]. CD8+ T cell expansion is reduced by the presence of NK cells and the depletion of NK cells leads to elevated CD8+ and CD4+ T cell frequencies. Interestingly, the depletion of both CD8+ T cells and NK cells leads to elevated EBV viral loads compared to depleting NK cells alone [19]. T cells also play an important role in controlling EBV tumorigenesis [16,47]. The depletion of CD8+ T cells results in EBV mediated tumorigenesis in HIS mice and elevated viral loads and the depletion of both CD4+ and CD8+ subsets resulted in further elevated viral loads and increased tumorigenesis [16]. While the interrogation of T cell responses during EBV infection is possible in HIS animals, the examination of B cell responses is still difficult. This is likely due to poor germinal center organization [30] and incomplete B cell maturation in HIS animals. Weak IgM and IgG responses have been observed in HIS animals following reconstitution [15,48,49], and EBV specific responses are largely lacking, with the exception of IgM production upon EBV infection [17]. Efforts to promote better germinal center organization and immunoglobulin class switching may generate superior B cell responses [26], and together with the use of MHC Class II transgenic animals may ensure more effective B cell/T cell cross talk.
Conclusions HIS mice are able to model certain important aspects of EBV infection and immune control. These include latency III and low levels of lytic EBV infection, LPD and cellmediated immune control by NK and T cells. Their main limitations are weak to absent humoral immune responses and a lack of routine genetic manipulations of the reconstituted human immune system compartments thus far. Furthermore, the compromised secondary lymphoid organ development in most currently used HIS mouse models, impedes not only isotype switched antibody responses, but also programs of EBV infection that might be dependent on germinal center reactions (latencies I and II) and also lymphocyte differentiation, like immature NK cell compartments that are enriched in these www.sciencedirect.com
EBV infection in HIS mice Gujer et al.
anatomical locations. However, if these challenges can be overcome, both immune responses and their protective function during infection with human specific pathogens like EBV can be efficiently studied and vaccine candidates can be explored for their capacity to induce these protective responses.
15. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A, Manz MG: Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 2004, 304:104-107.
Acknowledgements
17. Yajima M, Imadome K, Nakagawa A, Watanabe S, Terashima K, Nakamura H, Ito M, Shimizu N, Honda M, Yamamoto N, Fujiwara S: A new humanized mouse model of Epstein-Barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses. J Infect Dis 2008, 198:673-682.
CG is supported by the Marie Heim-Vo¨gtlin program of the Swiss National Science foundation (SNF; PMPDP3_145504) and DM by a fellowship awarded by the Swiss Academy of Medical Sciences and the SNF (323530_145247). Our laboratory is financed by grants to CM from Cancer Research Switzerland (KFS-3234-08-2013), Worldwide Cancer Research (14-1033), KFSPMS and KFSPHHLD of the University of Zurich, the Baugarten Foundation, the Sobek Foundation, Fondation Acteria, the Swiss Vaccine Research Institute and the SNF (310030_143979 and CRSII3_136241).
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