In this review, we will discuss the likelihood that a T cell response is elicited in the liver during the pre-erythrocytic phase, and the predicted outcome arising from such intrahepatic activation. Our arguments support a model in which sporozoites have developed strategies to induce tolerance but also reduce to a minimum the chances of encountering T cells by delaying efficient elimination of infected hepatocytes.

#### **SITES OF T CELL PRIMING DURING THE PRE-ERYTHROCYTIC PHASE**

Unless an individual has been previously infected with malaria parasites, their immune repertoire is predominantly naïve with regard to parasite-specific responses. Naïve T cells recirculation is restricted to the blood and lymph. Via these routes, these cells can also access secondary lymphoid tissues (spleen and LNs). In LNs, naive T cells scan for foreign antigens presented by dendritic cells (DCs) in the form of peptide-MHC (p:MHC) complexes. Primary T cell activation by DCs allows blast formation, cytokine secretion, proliferation, and differentiation into cells endowed with effector function and the capacity for trans-endothelial migration and entry into tissue parenchyma.

As the dermis is the site of inoculation and the first site exposed to sporozoites (**Figure 1**), it is not surprising that, as assessed by IFN-γ production, the first signs of T cell activation after mouse immunization with irradiated sporozoites (via mosquito bite or intradermal injection) were detected in the skin draining LNs at 48 h (Chakravarty et al., 2007). TCR transgenic T cells specific for the immunodominant antigen, the circumsporozoite protein (CSP), were activated only in these LNs, but effector T cells secreting IFN-γ were not detected in spleen, liver, or liverdraining LNs of the recipients at this time point (Chakravarty et al., 2007). Using a subcutaneous immunization protocol and CD81−*/*<sup>−</sup> mice that facilitate restriction of antigen presentation to the skin draining LN rather than the liver, it has been shown that T cell priming in the skin draining LN is sufficient to confer protection (Obeid et al., 2013). Depletion of CD11c+ cells abrogated efficient CD8 T cell priming (Jung et al., 2002; Obeid et al., 2013), supporting the model that DCs carrying the sporozoitederived antigen from the dermis migrated to the draining LNs, where they induced intranodal priming of CSP-specific T cells. Although there is strong evidence that the skin draining LNs prime T cells specific for early pre-erythrocytic proteins such as CSP, other sporozoite proteins are specifically expressed by hepatocytes during the subsequent liver stage. Furthermore, although CSP is an immunodominant antigen in Balb/c mice, it is not immunogenic for CTLs in C57Bl/6 mice, and in most humans naturally infected with malaria parasites (Gruner et al., 2007). Evidence that CSP is not the only protein that contributes to protection includes the development of protective immunity in mice tolerant for CSP that were immunized with irradiated sporozoites (Kumar et al., 2006), and the occurrence of CSP-independent sterile protection under chloroquine prophylaxis after immunization with irradiated sporozoites (Gruner et al., 2007; Mauduit et al., 2010). It is unlikely that the skin-draining LNs play any role in priming CD8 T cells against these mid to late pre-erythrocytic proteins expressed by hepatocytes.

A recent study (Lau et al., 2014) demonstrated that activated TCR transgenic CD8 T cells specific for an unidentified antigen expressed by both liver and blood stage parasites protected against subsequent *Plasmodium* infection, indicating that targeting antigens expressed by hepatocytes can lead to protective immunity. The compartment in which priming occurs is not clear. The liver draining LNs are the most likely site in which CD8 T cells can be primed following cross-presentation of hepatocyte-derived proteins by DCs. However, it is possible that proteins expressed by hepatocytes induce T cell activation and priming in the liver itself.

#### **THE LIVER: A SITE OF PRIMARY ACTIVATION FOR CD8 T CELLS**

The liver is now recognized as an alternative site (and the only non-lymphoid organ in the body) in which naive CD8 T cells can be activated independently of lymphoid tissues (Bertolino et al., 2001). First demonstrated for CD8 T cells, this remarkable hepatic immunological property has recently been extended to CD4 T cells (Tay et al., 2014b). Thus, in addition to the hepatic draining LNs, primary activation of T cells specific for mid to late pre-erythrocytic proteins could potentially occur in the liver itself. As proteins such as CSP are also expressed during the early phase of liver infection, intrahepatic tolerance might not only

affect naive T cells undergoing primary activation in this organ, but also alter the fate of anti-CSP effector T cells primed in skin draining LNs.

The ability of the liver to support primary CD4 and CD8 T cell activation is likely enabled by its unusual architecture and distinct resident populations of antigen-presenting cells.

#### **UNIQUE ARCHITECTURE OF THE LIVER**

The liver receives up to 25% of the cardiac output. Thus, T cells pass through this organ multiple times during their lives, and have the opportunity to interact with hepatic cells located in the sinusoids. The structure of the liver sinusoids is key to understand its role in intrahepatic T cell retention. Liver sinusoids are narrow capillaries with an average diameter of 10μm, in which blood flow is more intermittent and slower than in other capillaries. This unusual pattern of blood flow is contributed to by the narrow diameter of the sinusoids, the contraction of contractile smooth muscle sphincters in the walls of hepatic arterioles (Macsween and Scothorne, 1979; Macphee et al., 1995), and intermittent obstruction of the lumen by KCs (McCuskey and Reilly, 1993). This unique architecture favors interactions between leukocytes and hepatic cells, and intrahepatic recruitment of T cells recognizing their cognate antigen within this organ. Selectin-dependent leucocyte rolling normally observed in capillaries is not observed in liver sinusoids, and selectins are not required for the recruitment of leucocytes to this organ (Wong et al., 1997; Bowen et al., 2004). In contrast, TCR/p:MHC complex interactions and those between the adhesion molecules LFA-1 and ICAM-1 are important to the retention of lymphocytes, and these have been shown to play a critical role in the recruitment of CD8 T cells to the liver (Bertolino et al., 2005).

#### **POTENTIAL APCs IN THE LIVER SINUSOIDS**

Several hepatic cell types have been shown to be able to activate naive CD8 T cells *in vitro* (reviewed in Bertolino et al., 2002). It is now accepted that most liver APCs located in the sinusoids are able to induce primary activation *in vivo* as long as they can access circulating T cells and express or capture antigen.

*Hepatocytes* are located behind the endothelial cell layer, and are the principal cell type of the liver, comprising 75% of hepatic cells. These very large polyhedral, mono or bi-nucleated cells contain up to 16N DNA content, and synthesize and secrete a wide variety of biologically crucial molecules. They also metabolize and excrete many endogenous and xenobiotic substances. Steady state hepatocytes express MHC class I molecules (both classical and monomorphic CD1 molecules), and ICAM-1; inflammation also induces expression of MHC class II molecules, CD40L, and costimulatory molecules such as CD80 and CD86 (Bertolino et al., 1998; Holz et al., 2008). Since they normally lack MHC class II expression, resting hepatocytes may therefore only act as APCs for CD1d- and MHC class I-restricted T cells. Primary hepatocytes have also been demonstrated to drive efficient activation and proliferation of naïve TCR transgenic CD8 T cells *in vitro*, confirming their role as efficient APCs (Bertolino et al., 1998).

Despite the widespread view that hepatocytes are in a concealed location, express low levels of MHC class I, and therefore cannot be "seen" by T cells, available data suggests that, despite their sub-endothelial location, this cell population efficiently interacts with circulating CD8 T cells, and functions as effective APCs. *In vivo* interactions between hepatocytes and naïve CD8 T cells have been demonstrated or suggested in different animal models including transgenic mice (Bertolino et al., 2001; Bowen et al., 2004; Derkow et al., 2007), liver transplants (Klein and Crispe, 2006a; Tay et al., 2013), and mice treated with recombinant adeno-associated viral (rAAV) vectors (Wuensch et al., 2006, 2010; Tay et al., 2014a,b). Using electron microscopy, we have shown that circulating T cells make direct contact with hepatocytes through cytoplasmic extensions penetrating the endothelial fenestrations that perforate the LSECs (Warren et al., 2006). Furthermore, in contrast to early data, this study also demonstrated that interactions between T cells and liver parenchymal cells are maximized by the polarized expression of molecules required for primary T cell activation, MHC class I and ICAM-1, on the perisinusoidal cell membrane of hepatocytes (Warren et al., 2006).

*Liver sinusoidal endothelial cells* are efficient scavenger cells strategically located in the sinusoids, and able to clear low-density lipoprotein (LDL) and capture particulate antigens and immune complexes circulating via the blood and deliver them to hepatocytes (Sorensen et al., 2012). This uptake is mediated by mannose receptor, and has been shown to be more efficient than that mediated by KC, suggesting that LSEC specialize in this function. LSEC constitutively express both MHC class I and class II molecules, low levels of CD86, as well as adhesion molecules including ICAM-1, VCAM-1 and dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). They have been shown to activate both naïve CD4 and CD8 T cells *in vitro* (Knolle et al., 1999) and *in vivo* (Limmer et al., 2000).

*Kupffer cells (KC)* are the major population of tissue macrophages in the body, and are relatively heterogeneous (Ikarashi et al., 2013). Although some cells with KC-like properties can be reconstituted from bone marrow after lethal irradiation, most KC are derived from the yolk sac early after birth (Jakubzick et al., 2013). Intravital spinning disk and multiphoton microscopy experiments have demonstrated that KC are able to efficiently capture pathogens circulating within the sinusoids, such as *Borrelia burgdorferi* (Lee et al., 2010), and contribute to the formation of hepatic granulomas in *Leishmania donovani* (Beattie et al., 2010) and *Mycobacterium bovis* BCG (Egen et al., 2008) infections. KCs express MHC class I and II molecules, ICAM-1, CD86, CD80 and function as APCs for naïve CD8 T cells and CD4 T cells *in vitro* (Bertolino et al., 2002; Ebrahimkhani et al., 2011). *In vivo*, there is now data showing that liver bone marrow-derived cells (which include both KC and DC) are able to efficiently retain adoptively transferred naïve TCR transgenic CD8 T cells in an antigen-specific manner. This retention leads to the rapid activation of the T cells within the liver (Bowen et al., 2002; Holz et al., 2012; Tay et al., 2014b).

*Hepatic stellate cells (HSC),* also known as Ito cells, are located between LSEC and hepatocytes. Following liver damage and regeneration, they secrete the extracellular matrix that is critical for tissue repair. However, this process is also responsible for the development of fibrosis. In a non-inflamed liver, these cells play a role in regulating blood flow via constriction to modulate the diameter of the sinusoids (Rockey, 2001). Cultured HSC express MHC Class I and II, CD1d, and low levels of CD80 and CD86 (Vinas et al., 2003; Winau et al., 2007). Contact between HSC and T cells has not been directly observed; however, it is likely that they occur via LSEC fenestrations as demonstrated for hepatocytes (Warren et al., 2006). The role of these cells in intrahepatic T cell activation is still controversial. However, a key study has demonstrated that HSCs are also able to activate naïve CD8 T cells within the liver (Winau et al., 2007). In contrast, a more recent study suggested that HSC are poor T cell stimulators and function more as regulatory bystanders by promoting Tregs and suppressing Th17 differentiation (Ichikawa et al., 2011). These variant results might be due to contaminants in HSC preparations or to phenotypic changes after culture (Vinas et al., 2003). Thus, further *in vivo* data is required to clearly demonstrate the role of HSC as APCs in the intact liver.

It is important to note that the liver also contains DCs and biliary epithelial cells. However, these cell types are located within the portal tracts, behind a non-fenestrated endothelial cell layer, and there is no experimental evidence suggesting that they are able to initiate intrahepatic primary T cell activation (Holz et al., 2010b). In addition, hepatic DCs are mostly immature, suggesting that, like most tissue DCs, these cells induce primary T cell activation in liver draining LNs following maturation and migration triggered by inflammation.

#### **CROSS-PRESENTATION IN THE LIVER**

Antigen cross-presentation is a specialized function restricted to a limited number of APCs. In lymphoid tissues, only some DC subsets can perform this function. In the liver, besides liver resident CD8+ DCs, cross-presentation has been clearly demonstrated for LSEC, but the role of other hepatic APCs remains controversial.

The role of LSEC in cross-presenting antigen *in vivo* has been demonstrated in a series of *in vitro* and *in vivo* studies by Knolle and colleagues, clearly establishing these cells as the main crosspresenting cells of the liver. Primary activation of CD8 T cells by LSEC generally promotes tolerance (Limmer et al., 2000) via several mechanisms, including decreased expression of CD80 and CD86 molecules required for efficient priming by secretion of the immunosuppressive molecules PGE2 and IL-10 (Knolle et al., 1998), up-regulation of the inhibitory ligand PD-L1 (Diehl et al., 2008), and interaction with DC leading to loss of IL-12, CD80 and CD86 expression (Schildberg et al., 2008). However, LSECactivated CD8 T cells appear to survive *in vivo* for prolonged periods, and can be reactivated and rescued from deletion upon secondary encounter with DC (Bottcher et al., 2013).

The role of KC in cross-presenting antigen *in vivo* and the outcome of such activation are more controversial. *In vitro*, KC have been shown to inhibit T cell activation by secreting IL-10 (Knolle et al., 1995), but these cells also increase their expression of MHC class II and their APC function upon TLR3 ligation (Maemura et al., 2005; You et al., 2008). *In vivo*, CD8 T cell activation by KC leads to CD8 T cell apoptosis (Holz et al., 2012), suggesting that these APCs induce tolerance in the absence of inflammation. By comparing the ability of hepatocytes, LSEC, and KC to cross-present cell-associated antigen *in vitro*, Ebrahimkhani and colleagues have recently shown that LSEC are more efficient than KC in cross-presenting antigen (Ebrahimkhani et al., 2011).

Although one study also suggests that HSC can cross-present antigen (Winau et al., 2007) they have also been described as poor APCs and tolerogenic (Eksteen et al., 2009). HSC also appear to promote the expansion of FoxP3+ regulatory T cells (Jiang et al., 2008; Ichikawa et al., 2011) due to their high expression levels of retinoic acid and TGF-β (Schwabe et al., 2006).

The role of hepatocytes in cross-presentation remains controversial: although some studies have shown that purified hepatocytes cross-present cell-associated antigen *in vitro* (Ebrahimkhani et al., 2011), other studies (Limmer et al., 2000; Bongfen et al., 2007; Cockburn et al., 2011) suggest the opposite.

#### **INTERACTION OF SPOROZOITES WITH LIVER APCs**

After entering via the skin, sporozoites migrate to the liver where they glide along the hepatic sinusoids (**Figure 1**). The various cell populations located in the hepatic sinusoids are critical for the malaria sporozoites to establish infection in the liver.

HSC proteoglycans protruding through the endothelial fenestration into the sinusoidal lumen have been shown to be critical to the initial retention of circulating sporozoites (Frevert et al., 1993; Pradel et al., 2002). Real time intravital microscopy experiments have revealed that following retention, some sporozoites glide along the sinusoid till they interact with KC (Frevert et al., 2005). These cells express chondroitin and heparan sulfate proteoglycans that promote sporozoite arrest (Pradel et al., 2002). The apical cell pole of the parasite then positions itself against KC and after a pause, the sporozoite traverses the KC, pushes slowly across the sinusoidal cell barrier, and reaches the space of Disse (**Figure 1**) (Frevert et al., 2005). Whether the sporozoite enters via LSEC fenestrations or through the gap separating two adjacent LSEC has not been resolved, but the results of a recent study suggest that 25% of sporozoites can cross LSEC via the paracellular route (Tavares et al., 2013). Once in the space of Disse, sporozoites seem to increase their velocity and migrate for many minutes through several hepatocytes, before they eventually localize to a final one (Frevert et al., 2005) in which they establish a parasitophorous vacuole (Mota et al., 2001). Hepatocytes are required for the parasite to change its form and multiply. After a period of 7 days in humans for *P. falciparum* or 2 days in mice for *P. berghei/P. yoelii*, each infected liver cell releases 10,000–30,000 merozoites that will subsequently infect red blood cells (**Figure 1**). This classically marks the end of the silent pre-erythrocytic phase, and the beginning of the symptomatic erythrocytic stage of the infection (Kappe et al., 2003; Frevert, 2004; Heussler et al., 2006).

The gateway model, based on observations made using real time intravital microscopy, suggests that sporozoite migration from the sinusoidal lumen into the space of Disse require KC (Baer et al., 2007). Consistent with the critical role of KC, *P. yoelii* infection of mutant CSF-1-deficient *op/op* mice, that possess 75% less KC (and other macrophages), leads to an 80% decrease in the number of infected hepatocytes (Baer et al., 2007). It is not entirely clear why the parasite would require KC to cross the endothelium, and take the risk of triggering innate immunity, instead of directly crossing the endothelium. Interestingly, avian and reptilian *Plasmodium* species invade and replicate in KC and other macrophages instead of infecting hepatocytes. It has been postulated that during evolution in mammalian hosts the parasite has retained the ability to bind to KC, but gained the property of infecting hepatocytes instead of KC (Baer et al., 2007). Electron micrographs often show KC resting in the lumen of the sinusoids, anchored between two adjacent LSEC. For this reason, it has been proposed that KC act as a portal for sporozoites to cross the endothelial barrier and reach the parenchyma (Baer et al., 2007).

A more recent study using intravital laser spinning-disk confocal microscopy has however demonstrated that, although the majority (78%) of all sporozoite crossing events associated with cell-traversal activity involve KC, 22% involve direct cell traversal of the LSEC without involving a KC (Tavares et al., 2013). KC are thus not an absolutely mandatory gateway for sporozoite crossing of the hepatic sinusoidal barrier. Interestingly, this study also provided evidence that live sporozoites deficient in cell traversal activity were trapped and degraded in KC, while only ∼10% of control sporozoites (probably those that have exhausted their CTL capacity or alternatively those that are already dead before they arrive in the liver) displayed similar lasting interactions with KC. Although live sporozoites endowed with cell traversal ability are not trapped and killed by KC, they have been reported to inactivate and eventually kill the traversed macrophage resident cell (Klotz and Frevert, 2008). The authors of this study speculated that the main purpose of sporozoite cell traversal in the liver is to avoid parasite clearance (Tavares et al., 2013).

#### **ROLE OF THE LIVER IN PRIMARY T CELL ACTIVATION DURING THE PRE-ERYTHROCYTIC PHASE**

Due to the very low number of hepatocytes infected during natural transmission, and the short duration of the liver phase (between 2 and 7 days depending on the host species and the malaria parasite strain) (Overstreet et al., 2008), the role of the liver in priming CD8 T cells during natural transmission has been difficult to address. Some studies have suggested that the low dose of immunogen transmitted via one mosquito inoculum is not sufficient to induce a clonal burst sufficient to generate an effective T cell response (Hafalla et al., 2002). In addition, *Plasmodium* blood stage infection has been shown to inhibit the maturation and the capacity of DCs to initiate immune responses and suppress protective CD8 T cell responses against the liver stage *in vivo* (Ocana-Morgner et al., 2003). Although it does not necessarily extrapolate to natural *Plasmodium* transmission, immunization with high numbers of irradiated or genetically attenuated sporozoites that are capable of infecting hepatocytes, but do not develop in the liver, excludes any effect of blood stage parasites. This type of immunization allows a better understanding of the liver contribution to CD8 T cell priming and is critical in optimizing vaccine strategies.

Experiments performed using CSP-specific transgenic T cells adoptively transferred into mice immunized with irradiated or non-irradiated sporozoites argued against any significant effective CD8 T cell activation in the liver and liver draining LNs (Chakravarty et al., 2007). However, although this study excluded any contribution of the liver in priming protective anti-CSP CD8 T cells after *Plasmodium* infection, it did not rule out a role for the liver in T cell activation during malaria parasite infection for 2 main reasons. Firstly, T cell activation in this study was assessed by IFN-γ production at 48h instead of other functionally independent markers of T cell activation at earlier time points. Although a hallmark of efficient priming and effector CD8 T cell function, IFN-γ is poorly expressed by hepatocyte-activated CD8 T cells (Holz et al., 2008, 2012). In addition, the majority of T cells activated in the liver undergo rapid deletion, leading to tolerance (Holz et al., 2010a). Secondly, this study focused on CSP, a protein expressed by sporozoites before they invade hepatocytes. Expression of this protein decreases after invasion, while other proteins more specific for the liver stage of the parasite start to be expressed.

The role of the liver in priming T cells specific for mid to late pre-erythrocytic proteins therefore remains to be addressed. Genetically attenuated sporozoites, designed to have arrested development at a late liver stage, induced larger and broader CD8 T cell responses compared to radiation attenuated sporozoite or early-arresting genetically attenuated parasite immunizations (Butler et al., 2011). They also promoted superior protection in inbred and outbred mice (Butler et al., 2011), providing further support for the idea that bona-fide liver stage antigens can prime protective immune responses.

A recent study by Michael Bevan and colleagues illustrates why this question is important for anti-malaria parasite vaccination. By using high-throughput screening and irradiated or attenuated sporozoites, these investigators identified a unique CTL response against an epitope derived from the parasite ribosomal L3 antigen (Murphy et al., 2013), a protein expressed during the liver-stage and in erythrocytes. Surprisingly, these CTLs did not follow the general pattern of anti-CSP CTL responses, as they could be expanded by heterologous prime-boost regimens, but not by multiple sporozoite immunizations. These findings illustrate that CTL responses specific for liver-stage proteins might require different immunization strategies than those specific for antigens expressed by the incoming parasite. Although this study did not clarify the mechanisms responsible for these distinct CTL patterns or whether other liver-stage proteins generate similar patterns, it is tempting to speculate that they are caused by different priming conditions in the liver and LNs.

Several studies have shown that intravenous administration of attenuated sporozoites is superior to other routes in conferring protection (Spitalny and Nussenzw, 1972; Richards, 1977; Chatterjee et al., 1999; Douradinha et al., 2007; Epstein et al., 2011) As sporozoites traveling via the intravenous route reach the liver, these experiments support a major contribution of this organ in priming effector cells. A recent study reporting the results of a clinical trial of an anti-malaria parasite vaccination on 80 volunteers, confirmed that intravenous administration of purified irradiated sporozoites promoted protection, while inoculation of the vaccine via the skin was suboptimally immunogenic and protective (Epstein et al., 2011). A high frequency of sporozoite-specific IFN-γ–producing CD8 T cells in the liver was shown to confer protection in mice (Epstein et al., 2011). Although it does not demonstrate the direct role of hepatic cells in priming effector T cells during natural transmission, this study suggests that targeting the liver route of activation might promote protection.

Presentation of liver-stage sporozoite proteins to antigenspecific CD8 T cells can occur either directly by infected hepatocytes (direct MHC Class I presentation pathway), or indirectly by APCs that capture extracellular sporozoite-derived proteins synthesized by hepatocytes and present them in the context of MHC I molecules (cross-presentation pathway). These two pathways are detailed below.

#### **ROLE OF INFECTED HEPATOCYTES IN PRIMARY T CELL ACTIVATION IN MALARIA**

Although sporozoites interact with many liver cell types, experiments using bone marrow chimeric mice have demonstrated that radio-resistant cells (most likely to be infected hepatocytes) are likely to be the main cells presenting antigens derived from irradiated sporozoites to effector CD8 T cells in the liver (Chakravarty et al., 2007). Furthermore, it has been shown that while DCs cross-present CSP via endosomes, presentation of CSP by hepatocytes requires expression of the protein in the cytosol rather than in endosomes (Cockburn et al., 2011). CSP presentation by hepatocytes was independent of cross-presentation (Cockburn et al., 2011). Collectively, these results provide strong evidence that hepatocytes are the main liver cells infected by irradiated sporozoites, and able to present cytosolic sporozoite antigens to T cells via the direct MHC class I presentation pathway.

Some evidence also suggests that replication-competent malaria parasite-infected hepatocytes contribute to CD8 T cell priming: (i) elimination of hepatic stage parasites abrogates protection generated by administration of irradiated sporozoites (Scheller and Azad, 1995); and (ii) protection induced by live sporozoites in mice treated with chloroquine was abrogated by primaquine treatment that eliminates the parasite from hepatocytes (Belnoue et al., 2004).

Most studies investigating the early immune events in malaria have used mice immunized with a large dose of irradiated sporozoites, and have focused on the abundant CSP that covers the surface of sporozoites. This protein has multiple functions in several stages of the parasite life cycle. It is thought to be a master regulator of the development of the pre-erythrocytic stages (Singh et al., 2007), and inhibits KC immune defense (Usynin et al., 2007). In infected hepatocytes, CSP traverses the membrane of the parasitophorous vesicle, enters the cytoplasm, and translocates to the nucleus where it downregulates expression of the transcription activation factor NF-κB (Singh et al., 2007). This inhibits host inflammatory immune functions, and promotes the development of the liver stage parasite (Singh et al., 2007). Paradoxically, by entering the cytoplasm, CSP exposes itself to proteasomal degradation and processing by the MHC Class I presentation pathway. CSP has been shown to be an immunodominant protective T cell antigen (Kumar et al., 2006). Mouse primary hepatocytes have also been shown to be able to process *Plasmodium berghei* CSP after live sporozoite infection, and present CSP-derived peptides to specific CD8 T cells *in vitro* in a proteasome-dependent manner (Bongfen et al., 2007). A recent study (Ma et al., 2013) performed using human primary hepatocytes has also confirmed that the direct MHC class I presentation pathway is not markedly affected by live *P. berghei* sporozoites: mRNA expression levels of all major components of the MHC class I processing and presentation machinery were not significantly affected by parasite replication at 24 or 48 h post-infection, while the levels of MHC class I expression were decreased only by 10–20% at the surface of infected host cells as compared to either uninfected or untreated controls. Furthermore, ectopic expression of CSP did not interfere with basal MHC class I expression, or IFN-γ and TNF-α-induced upregulation of MHC class I expression (Ma et al., 2013).

These results therefore imply that CSP translocation to the cytoplasm increases the risk of recognition and elimination of infected hepatocytes following presentation of CSP-peptides by the classical MHC class I presentation pathway. More recently, LISP-2, a protein specifically expressed by mid to late stage parasites, has also been shown to be exported to the cytosol from the parasitophorous vacuole (Orito et al., 2013). If this protein is immunogenic, it also have the potential to be an immune target.

It is thought that the important function of CSP in the parasite development justifies why the parasite exposes itself to the immune system in such a way. An alternative view is that primary activation by hepatocytes leads to tolerance, and therefore this is a low risk strategy for the parasite. Expression of CSP and other liver-stage specific proteins on liver cells might also be critical to tolerize effector CD8 T cells primed in skin-draining LNs.

#### **OUTCOME OF PRIMARY CD8 T CELL ACTIVATION BY HEPATOCYTES**

Hepatocytes are not "professional" APCs as they do not express known costimulatory molecules such as CD80 and CD86. Although these cells are able to induce primary activation and proliferation of CD8 T cells, it is expected that this activation would not be as efficient as priming induced by mature DCs. Our initial *in vitro* work demonstrated that naive CD8 T cells co-cultured with antigen-expressing hepatocytes were activated, proliferated, and started to acquire cytotoxic activity, but died prematurely by apoptosis (Bertolino et al., 1998, 1999), suggesting that hepatocytes promoted T cell deletion and tolerance.

To investigate the fate of CD8 T cells activated by antigenexpressing hepatocytes *in vivo*, we have used transgenic models and rAAV vectors to restrict antigen or relevant p:MHC expression to these cells *in vivo*. Hepatocyte-activated T cells adoptively transferred into mice expressing the cognate p:MHC complex on all hepatocytes were deleted within the first 4 days post-activation, without developing significant cytotoxic activity (Bertolino et al., 2001; Bowen et al., 2004; Holz et al., 2008; Tay et al., 2014a), suggesting that this activation induced tolerance. In these studies, most CD8 T cells were rapidly deleted within the first few hours post-activation as a result of invading hepatocytes and subsequent degradation inside lysosomal compartments (Benseler et al., 2011). Invasion of a cell into another cell is known as "emperipolesis" (Humble et al., 1956; Overholtzer and Brugge, 2008), and is observed in liver sections in autoimmune and viral hepatitis (Dienes, 1989). To distinguish this process of CD8 T cell deletion within hepatocytes from emperipolesis without destruction of the invading cell, we have termed this process "suicidal emperipolesis" (Benseler et al., 2011).

Some T cells surviving suicidal emperipolesis proliferate, but subsequently die as a result of primary activation in the absence of costimulatory molecules. This "death by neglect" has been shown to be associated with low expression of cytokines and high expression of the pro-apoptotic molecule Bim (Holz et al., 2008).

Although hepatocytes generally induce T cell tolerance *in vivo*, recent studies have suggested that this is not always the case. In some experimental systems, CD8 T cells activated by hepatocytes survived and became full effector cells (Wuensch et al., 2006; Klein and Crispe, 2006b; Derkow et al., 2007). Our results suggest that generation of CTLs by hepatocytes requires high TCR affinity interactions and low frequencies of antigen-expressing hepatocytes; in the absence of these requirements, CD8 T cell tolerance ensues in response to hepatocyte expressed antigen (Tay et al., 2014a).

In malaria parasite infection, the frequency of sporozoiteinfected hepatocytes is low. Our recent studies therefore predict that infected hepatocytes should be able to prime at least some high affinity sporozoite antigen-specific T cells and generate full effector cells that contribute to the anti-malaria CTL pool.

#### **ROLE OF CROSS-PRESENTING APCs IN THE ANTI-MALARIA RESPONSE**

Unless the parasite has developed strategies to inhibit this process, it is very likely that following entry into hepatocytes, some antigens are shed, captured, and cross-presented by other cell types in lymphoid tissues and/or the liver (**Figure 1**).

Although liver DCs are not accessible to naive T cells and do not therefore contribute to intrahepatic activation, it is likely that these APCs play a major role in cross-presenting antigens in the liver draining LNs. In studies of transgenic models and using the rAAV system mentioned above, our results suggest that cross-presentation of antigens expressed by hepatocytes leads to efficient T cell priming, resulting in full effector function (Tay et al., 2014a). We have postulated that antigen presentation in lymphoid tissues is the main pathway facilitating the generation of CTLs directed against liver pathogens (Bowen et al., 2004, 2005).

Until recently, the exact location of the liver-draining LNs in mice had not been identified (Barbier et al., 2012). Although likely, the contribution of these lymph nodes to CTL generation during natural malaria parasite infection has not been clearly demonstrated. However, a recent study (Lau et al., 2014) in which mice were intravenously injected with high numbers of irradiated sporozoites demonstrated preferential malarial-antigen specific CD8 T cell activation and proliferation in the 3 hepatic draining LNs (Barbier et al., 2012). The number of intrahepatic CD11c+ cells has also been reported to increase in mice injected with irradiated *P. yoelii* sporozoites or from live parasites inoculated by infected mosquitoes (Leiriao et al., 2005b). These potential APCs have been shown to contain intracellular hepatocyte proteins, suggesting that debris of apoptotic hepatocytes were taken up by liver DCs (or recruited circulating DCs or monocytes) (Leiriao et al., 2005b). Whether these cells migrate to the draining LNs and cross-present sporozoite antigens to T cells has not been investigated in this study. Another study (Jobe et al., 2009) used irradiated sporozoites to demonstrate that conventional CD8+ DC able to induce the differentiation of naïve CD8 T cells into CD44hiCD45RBloCD62Llo effector memory T cells accumulated in the livers of sporozoite-immunized mice, suggesting that this liver DC subset is involved in the induction of liver-stage antigen-specific CD8 T cells.

It is important to emphasize that most of these studies have used high numbers (5 <sup>×</sup> <sup>10</sup><sup>4</sup> to 1 <sup>×</sup> <sup>10</sup>6) of irradiated parasites injected intravenously, or mice bitten by 50 mosquitoes, that do not reflect natural transmission. Thus, although DCs might capture and cross-present sporozoite proteins from infected hepatocytes in the liver and/or draining LN when high numbers of sporozoites are administered, the role of DCs in cross-presenting sporozoite antigens derived from hepatocytes in the liver draining LNs during natural transmission requires further investigation.

The function and fate of anti-malaria T cells activated by potential cross-presenting hepatic APCs is relatively unknown. Experiments performed using radiation-induced bone marrow chimeras (Chakravarty et al., 2007) excluded any role for bone marrow–derived hematopoietic-lineage liver cells, such as bloodderived DCs or monocyte-derived macrophages, in the presentation of irradiated and live sporozoite-derived antigens to effector CD8 T cells, suggesting that these APCs did not present antigens to T cells. It is important to note that, despite the conclusions of the study, these experiments did not exclude a role for KC, as a large proportion of these liver-resident macrophages are radio-resistant (Kennedy and Abkowitz, 1997; Klein et al., 2007). In addition, the role of radio-resistant LSEC and stellate cells has not been clarified. Thus, in the absence of further experimental evidence, any discussion on this topic remains speculative.

It is clear that the parasite avoids being detected by CTLs primed in the skin draining LNs by changing expression of proteins after settling in hepatocytes. Once in the liver, it is likely that it uses other strategies to avoid being eliminated by CD8 T cells specific for liver stage proteins.

#### **COULD SPOROZOITES HARNESS THE TOLEROGENIC PROPERTY OF THE LIVER TO SUBVERT INTRAHEPATIC IMMUNITY AND PREVENT ELIMINATION OF INFECTED CELLS?**

The first model is that malaria sporozoites prevent the generation of efficient CTLs that would kill infected cells by inducing tolerance (*"subversion from within"* model, see **Figure 2**). This possibility is more relevant to infection by irradiated sporozoites that do not induce long lasting responses and could be tolerized.

Many studies in the literature suggest that the liver can tolerize effector T cells. In transplantation, an hepatic allograft can reverse rejection of previously transplanted organs from the same donor (Benseler et al., 2007), even when the liver was transplanted up to 6 days after the initial transplant, indicating that the liver allograft is able to induce tolerance in the recently activated T cell compartment. More recently, we have shown that expression of an allo-MHC class I molecule in most hepatocytes prevented the rejection of skin grafts expressing this allo-MHC molecule despite prior sensitization of recipient mice (Cunningham et al., 2013). Finally, analyses performed in transgenic mouse models indicated that TCR transgenic T cells activated in the LNs of recipient mice accumulated in the antigen-expressing liver, killed some hepatocytes, but never induced chronic liver damage, again suggesting that these effector T cells were silenced (Holz et al., 2010b, 2012).

How does the liver tolerize effector T cells? The original "graveyard" model proposed by Crispe and colleagues (Huang et al., 1994; Mehal et al., 1999) suggested that the liver was a disposal site for T cells activated in lymphoid tissues. This was consistent with several studies showing efficient intrahepatic retention of activated CD8 and CD4 T cells. The same group later proposed that the liver actively killed effector T cells recognizing their antigen within the liver. However, as this theory could not account for the presence of effective immune responses in the liver under some circumstances, and did not allow for the presence of high numbers of effector/memory T cells in this organ (Belz et al., 1998; Keating et al., 2007), this concept was subsequently abandoned (Crispe et al., 2006; Bertolino et al., 2007).

We have recently revisited this question by showing that the frequency of antigen-expressing hepatocytes is critical in determining tolerogenic outcome. When at least 25% of hepatocytes expressed antigen, effector T cells primed in lymphoid tissues were eventually silenced, while below this threshold cytotoxic T cells survived after clearing antigen-expressing hepatocytes (Tay et al., 2014a). This might explain the outcome of tolerance in transplantation and transgenic systems, where there is ubiquitous expression of antigen within the liver, and detection of memory T cells in some viral models (Belz et al., 1998; Keating et al., 2007).

The mechanisms by which the liver silences effector CD8 T cells in the presence of high dose antigen is not entirely clear, and molecules such as Galectin-1, TNFR, and FasL on hepatic cells have been suggested to play a key role in this process (reviewed in

Holz et al., 2010b, 2012). Our results suggest that most effector T cells surviving the early phases of activation expressed high levels of the pro-apoptotic molecule Bim, and died by apoptosis. T cells that survived this phase displayed poor CTL function, associated with an exhausted phenotype, including expression of high levels of the inhibitory molecule PD-1, as well as reduced TCR and CD3 expression (Holz et al., 2010b, 2012; Tay et al., 2014a), indicative of prolonged antigen exposure and reduced T cell activity (Ferber et al., 1994). It is also possible that suicidal emperipolesis plays an important role in the clearance of effector T cells recognizing their cognate antigen within the liver. This question is currently under investigation in our laboratory.

The extremely low frequency of infected hepatocytes during the pre-erythrocytic stage (even when a high number of irradiated sporozoites is used for immunization) is far from the high antigen dose promoting silencing of effector cells. Based on this argument, we predict that the few infected hepatocytes and potential cross-presenting hepatic APCs should be killed by CTLs, rather than inducing tolerance.

#### **DO SPOROZOITES AVOID BEING DETECTED BY CTLs?**

The alternative model to explain how malaria sporozoites use the liver to avoid being eliminated by the immune system is to hide in this large organ to minimize as much as possible the probability of encountering CTLs *("hide-and-seek" model,* see **Figure 3**). This model would apply to both live and irradiated sporozoites. From the parasite perspective, it might be efficiently achieved by infecting the minimum number of hepatocytes and by spending the shorter possible time in the host cell.

Using quantitative PCR assays, it has been estimated that a mosquito bite inoculates between 0 and 1297 live sporozoites into the skin, with a mean number of 123 and a median number of 18 (Medica and Sinnis, 2005). As some of these parasites remain in the skin or migrate to the LNs, only a few will make it to the liver (Sidjanski and Vanderberg, 1997; Jin et al., 2007). As a result, an extremely low number of hepatocytes become infected by live sporozoites. It is estimated that in mice, 1 in a million hepatocyte is infected while in humans the frequency is even lower (one in a billion) (Van Braeckel-Budimir and Harty, 2014).

It is not clear whether T cells specific for the liver-stage malaria parasite efficiently find the very few infected hepatocytes during the short period of time they reside in the liver. It is likely that they do not. This is particularly true for naive T cells for primary activation, but it would also be difficult for effector T cells to find their targets (secondary activation). As the liver is made of hundreds of millions of hepatocytes, this is akin to finding a needle in a haystack. Thus, the low number of infected hepatocytes combined with the short amount of time sporozoites reside in the cell might be a strategy used by the parasite to avoid both T cell priming in the liver and escape immune surveillance by **FIGURE 3 | Different potential strategies used by sporozoites to evade immune surveillance by CTLs according to the "hide and seek" model.** To avoid being eliminated by CTLs generated in the LNs or intrahepatically, malaria sporozoites might have developed several strategies: (i) the very low frequency of sporozoites lowers the probability of encountering CTLs; (ii) the

as a decoy to "buy" some precious time for the sporozoites.

CTLs. This might explain the apparent lack of effective immune response during the pre-erythrocytic stage and the lack of long lasting protection induced by both live and irradiated sporozoites. Consistent with this model, high numbers (105) of irradiated sporozoites injected intravenously did not elicit a very significant activation and proliferation of naive TCR transgenic CD8 T cells in the liver during the first 2 days in comparison to the spleen and liver draining LNs, although these T cells recognized an antigen expressed by hepatocytes (Lau et al., 2014). Effector cells generated in lymphoid tissues recirculated, however, to the liver at day 3 (Lau et al., 2014). These findings suggest that, in vaccination protocols using high numbers of irradiated sporozoites, most of the primary T cell activation occurs in lymphoid tissues rather than the liver. Whether this also occurs during natural transmission (lower physiological numbers of live sporozoites inoculated by mosquitos) requires, however, further demonstration. Regardless of whether they are generated in LNs or spleen, to achieve protective immunity CTLs would need to detect and kill all infected hepatocytes during the very short period of the preerythrocytic phase (2–7 days). One can predict that the success of this killing is likely to be critically dependent on the total number of effectors.

Results obtained using mouse and human immunization protocols using both attenuated or live sporozoites suggest that this assumption is correct. These protocols can only achieve protection by injecting 1000-fold or more sporozoites than occurs in natural transmission and/or by boosting the number of CSPspecific CD8 T cells (Overstreet et al., 2008). Immunization experiments in mice have suggested that long-term protection requires a remarkably strong CD8 T-cell response, representing a substantial fraction of the total CD8 T-cell pool (Schofield et al., 1987; Weiss et al., 1988; Romero et al., 1989; Schmidt et al., 2008). Optimal CD8 T cell priming was associated with persistence of CSP in both lymphoid tissues and livers of mice, and was mediated by DCs or macrophages (Cockburn et al., 2010). By investigating the CD8 T-cell immune response in Balb/c mice immunized with DC loaded with the immunodominant CSP, followed by a boost provided by *Listeria monocytogenes* expressing CSP, Harty and colleagues have recently shown that stable long term protection upon live sporozoite challenge required CSPspecific CD8 T-cell frequencies exceeding a threshold of 1% of total PBLs (Schmidt et al., 2008). This threshold depends on the mouse MHC strain and *Plasmodium* species (Van Braeckel-Budimir and Harty, 2014). However, it significantly exceeds

frequencies of antigen-specific CD8 T cells required for plausible protection against various viral and bacterial infections.

In addition to minimizing the probability of being recognized by T cells whilst within host hepatocytes, it is tempting to speculate that the sporozoite has developed several strategies to gain precious time and further decrease this probability. For rodent malaria parasites (*P. berghei* and *P. yoelii)*, the timing of T cell activation might be sufficient to avoid immune recognition by CTLs activated in LNs: by the time CTLs leave the LNs and circulate to the liver (2–3 days), merozoites would have been generated and the liver phase would be over. The pre-erythrocytic phase of *P. falciparum* in humans is longer (7–9 days) and other strategies might be used by the pathogen to evade or delay immune detection.

One of these potential strategies is to use a decoy to divert the T cells to non-infected cells. When live sporozoites traverse through several hepatocytes before establishing the parasitophorous vacuole in a selected host cell (Frevert et al., 2005), they disrupt the plasma membrane of several other hepatocytes (Mota et al., 2001). During this process, they leave a trail of CSP (and possibly other proteins) in the cytosol of affected cells (**Figures 1**, **3**). It is not clear whether hepatocytes recover from this cell traversal. Studies using high number of live parasites have detected many necrotic hepatocytes and increases in serum ALT (Frevert et al., 2005), suggesting that sporozoites cause hepatocellular injury. Whether this is an experimental artifact due to the high number of parasites, or whether it occurs during natural malaria transmission needs to be clarified. However, if some hepatocytes quickly repair their perforated membranes, it is likely that the CSP contained in the cytosol of non-infected cells will be processed by the MHC class I presentation pathway and form p:MHC complexes. Consistent with this, by using live *P. berghei* sporozoites deficient for the SPECT protein that is essential for cell traversal, Bongfen et al. (2007) have demonstrated that both infected and traversed primary hepatocytes process and present CSP to CD8 T cells. The processing and presentation pathway involved the proteasome, antigen transport through a post-endoplasmic reticulum compartment, and aspartic proteases (Bongfen et al., 2007).

Non-infected hepatocytes presenting CSP might be used by the sporozoite as a decoy strategy to divert CD8 T cells toward non-infected hepatocytes. This tactic might delay the time for T cell detection, and increase the chance of survival of infected hepatocytes. Interestingly, some reports suggested that infected hepatocytes become resistant to apoptosis (Leiriao et al., 2005a; Van De Sand et al., 2005), conferring an additional selective advantage to infected cells.

The reason why the sporozoite needs to traverse several hepatocytes before settling in one cell located at a distant site from the point of entry is also intriguing and could be an additional strategy developed by the parasite to further reduce the risk of being detected by T cells (**Figure 3**). Upon KC traversal, the abundant CSP on the sporozoite surface has been shown to induce cyclic AMP (cAMP) that inhibits the assembly of the NADPH oxidase (Usynin et al., 2007).  Although this prevents the parasite from being eliminated by the macrophage defense system while it crosses the endothelium, it is possible that the traversed KC releases cytokines and chemokines able to attract immune cells. This might explain why in addition to blocking the respiratory burst, viable sporozoites also generate an anti-inflammatory cytokine secretion profile in KC (Klotz and Frevert, 2008). KC exposed to infectious sporozoites down-modulated MHC class I, secreted no IL-12p40, and had reduced antigen presenting function (Steers et al., 2005), suggesting that the sporozites minimize the potential signals that KC might release to attract immune cells. Interestingly, in contrast to KC from naive mice exposed to infectious sporozoites, KC from irradiated sporozoite-immunechallenged mice up-regulated class I and costimulatory molecules and produced elevated IL-12p40 relative to naive KC. These KC also exhibited augmented APC activity (Steers et al., 2005).

Settling in the first hepatocyte at the initial entry point would be highly risky for the parasite, as CD8 T cells would rapidly find and kill the infected cell located close to the signaling KC. Traveling to a distant site might buy precious time for the parasite while it differentiates at a distant location.

In summary, malaria sporozoites seem to have developed several strategies to avoid killing of infected hepatocytes by sporozoite-specific CTLs. These include changing expression of proteins within the infected hepatocyte, infecting an organ where primary activation of CD8 T cells is biased toward the induction of tolerance, and developing several tactics to decrease the probability that infected hepatocytes encounter antigen-specific T cells. These arguments support the view that the predominant limitation for malaria-specific T cells is finding/eliminating all infected hepatocytes during the short period of the hepatic cycle phase, rather than overcoming tolerance in the liver.

Thus, although both malaria sporozoites and hepatotropic viruses target the liver, these pathogens seem to use totally different strategies to escape immune recognition. Instead of infecting many hepatocytes in a short period of time and exploiting the tolerogenic properties of the liver, malaria sporozoites infect very few cells, and have developed additional strategies to decrease the probability of being detected by CTLs. This model predicts that to be effective, anti-malaria vaccination protocols should aim to boost the number of CTLs to increase their probability of finding and killing all infected hepatocytes.

#### **ACKNOWLEDGMENTS**

The authors would like to thank William Heath, Michelle Vo and Yik Chun Wong for critically reading this review. This work was supported by the NHMRC Australia (Program grant 571408).

#### **REFERENCES**

hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells. *PLoS Pathog.* 6:e1000805. doi: 10.1371/journal.ppat.1000805

optimal CD8+T cell responses against malaria liver stage parasites. *PLoS Pathog.* 6:e1000877. doi: 10.1371/journal.ppat.1000877

You, Q., Cheng, L., Kedl, R. M., and Ju, C. (2008). Mechanism of T cell tolerance induction by murine hepatic Kupffer cells. *Hepatology* 48, 978–990. doi: 10.1002/hep.22395

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 20 October 2014; accepted: 12 January 2015; published online: 18 February 2015.*

*Citation: Bertolino P and Bowen DG (2015) Malaria and the liver: immunological hide-and-seek or subversion of immunity from within? Front. Microbiol. 6:41. doi: 10.3389/fmicb.2015.00041*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2015 Bertolino and Bowen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Corrigendum: Malaria and the liver: immunological hide-and-seek or subversion of immunity from within?

Patrick Bertolino\* and David G. Bowen

*Liver Immunology Group, Centenary Institute and AW Morrow Gastroenterology and Liver Centre, University of Sydney and Royal Prince Alfred Hospital, Sydney, NSW, Australia*

Keywords: LSEC, malaria, sporozoites, CD8 T cells, tolerance

#### **A corrigendum on**

**Malaria and the liver: immunological hide-and-seek or subversion of immunity from within?** by Bertolino, P., and Bowen, D. G. (2015). Front. Microbiol. 6:41. doi: 10.3389/fmicb.2015.00041

In the original article on page 4, there is an error in the following sentence:

#### Edited by:

*Ute Frevert, New York University School of Medicine, USA*

#### Reviewed by:

*Xun Suo, China Agricultural University, China Hajime Hisaeda, Gunma University, Japan*

> \*Correspondence: *Patrick Bertolino<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology*

Received: *31 March 2015* Accepted: *28 April 2015* Published: *13 May 2015*

#### Citation:

*Bertolino P and Bowen DG (2015) Corrigendum: Malaria and the liver: immunological hide-and-seek or subversion of immunity from within? Front. Microbiol. 6:460. doi: 10.3389/fmicb.2015.00460*

"[...] Liver sinusoidal endothelial cells are efficient scavenger cells strategically located in the sinusoids and able to clear low-density lipoprotein (LDL) and capture particulate antigens and immune complexes circulating via the blood and deliver them to hepatocytes (Sorensen et al., 2012). This uptake is mediated by mannose receptor, and has been shown to be more efficient than that mediated by KC, suggesting that LSEC specialize in this function. [...]"

The correct sentence is:

"[...] Liver sinusoidal endothelial cells are efficient scavenger cells strategically located in the sinusoids, and able to clear oxidised or acetylated low-density lipoprotein (LDL) and capture small particles (<200 nm) circulating via the blood (Sorensen et al., 2012). This uptake is mediated by several receptors and has been shown to be more efficient than that mediated by KC, suggesting that LSEC specialize in this function. [...]"

#### Funding

This work was supported by the NHMRC Australia (Program grant 571408).

#### References

Sorensen, K. K., McCourt, P., Berg, T., Crossley, C., Le Couteur, D., Wake, K., et al. (2012). The scavenger endothelial cell: a new player in homeostasis and immunity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 303, R1217–R1230. doi: 10.1152/ajpregu.00686.2011

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Bertolino and Bowen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Antigen-driven focal inflammatory death of malaria liver stages

#### **Ganchimeg Bayarsaikhan1,2 , Masoud Akbari <sup>2</sup> , Katsuyuki Yui <sup>2</sup> and Rogerio Amino<sup>1</sup>\***

<sup>1</sup> Unité de Biologie et Génétique du Paludisme, Département Parasites et Insectes Vecteurs, Institut Pasteur, Paris, France

<sup>2</sup> Division of Immunology, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

#### **Edited by:**

Ute Frevert, New York University School of Medicine, USA

#### **Reviewed by:**

Ute Frevert, New York University School of Medicine, USA Ann-Kristin Mueller, University Hospital Heidelberg, Germany Volker T. Heussler, University of Bern, Switzerland

#### **\*Correspondence:**

Rogerio Amino, Unité de Biologie et Génétique du Paludisme, Département Parasites et Insectes Vecteurs, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France e-mail<EMAIL_ADDRESS>
Multiple immunizations using live irradiated sporozoites, the infectious plasmodial stage delivered into the host skin during a mosquito bite, can elicit sterile immunity to malaria. CD8<sup>+</sup> T cells seem to play an essential role in this protective immunity, since their depletion consistently abolishes sterilizing protection in several experimental models. So far, only a few parasite antigens are known to induce CD8<sup>+</sup> T cell-dependent protection, but none of them can reach the levels of protection afforded by live attenuated parasites. Systematic attempts to identify novel antigens associated with this efficient cellular protection were so far unsuccessful. In addition, the precise mechanisms involved in the recognition and elimination of parasitized hepatocytes in vivo by CD8<sup>+</sup> T cells still remain obscure. Recently, it has been shown that specific effector CD8<sup>+</sup> T cells, after recognition of parasitized hepatocytes, recruit specific and non-specific activated CD8<sup>+</sup> T cells to the site of infection, resulting in the formation of cellular clusters around and in the further elimination of intracellular parasites. The significance of this finding is discussed in the perspective of a general mechanism of antigen-dependent focalized inflammation and its consequences for the elimination of malaria liver stages.

**Keywords: CD8**<sup>+</sup> **T cells, inflammatory infiltrate, cooperative behavior, in vivo imaging, Plasmodium**

#### **THE PRE-ERYTHROCYTIC PHASE OF MALARIA INFECTION**

Malaria is a mosquito borne disease caused by *Plasmodium spp.* parasites. Despite advances in control and prevention measures, malaria still kills > 600,000 people annually and no effective licensed vaccine is available so far (www.who.int/malaria/en). The disease is a consequence of repeated cycles of parasite invasion and replication inside red blood cells (RBC). However, before infecting the blood and causing the disease, the parasite must pass through a silent and asymptomatic pre-erythrocytic (PE) phase. In mammals, the PE phase starts with the inoculation of sporozoites into the extravascular regions of the host skin during a mosquito bite. Some of these highly motile stages get access to the blood circulation and home to the liver, where they traverse several hepatic cells before invading and developing, as liver stages, inside hepatocytes. One infected hepatocyte generates thousands of RBC-infective stages in 2 to ∼10 days depending on the species. Finally, the PE phase finishes with the release of these invasive stages into the blood circulation (Ménard et al., 2013).

In contrast to the symptomatic erythrocytic phase of infection, which can reach the magnitude of ∼10<sup>12</sup> circulating infected RBCs in hyperparasitemic adults (World Health Organization [WHO], 1990), the asymptomatic PE stages represent the smallest parasite burden (1∼1000 sporozoites and liver stages) inside the mammalian host (Medica and Sinnis, 2005). Consequently, these stages are considered as ideal targets for vaccine intervention, since early elimination of this minute population of extracellular sporozoites and intracellular liver stages could strategically block infection before pathogenesis and transmission of parasites to mosquitoes. Most importantly, immunizations using live sporozoites, which are blocked during hepatic development as consequence of irradiation (Nussenzweig et al., 1967), genetic modification (Mueller et al., 2005) or drugs (Friesen and Matuschewski, 2011), confer sterile protection against sporozoite re-infection in several experimental models, as well as in humans (Seder et al., 2013).

#### **THE PUZZLE OF LIVER STAGE KILLING BY CD8**<sup>+</sup>**T CELLS**

Although there is evidence that antibodies and CD4<sup>+</sup> T cells contribute to the protection induced by live irradiated sporozoites (Schofield et al., 1987; Tsuji et al., 1990; Rodrigues et al., 1993; Doolan and Hoffman, 2000), CD8<sup>+</sup> T cells seem to be the major players of this sterilizing immunity since in almost all tested rodent (Schofield et al., 1987; Doolan and Hoffman, 2000; Schmidt et al., 2010) and primate (Weiss and Jiang, 2012) models, sterile protection is abolished when CD8<sup>+</sup> T cells are depleted before sporozoite challenge. Accordingly, the transfer of parasitespecific CD8<sup>+</sup> T cells can also protect mice from sporozoite infection (Romero et al., 1989; Weiss et al., 1992).

This protective cellular response is associated with a high number of specific CD8<sup>+</sup> T cells circulating in the peripheral blood of protected mice, ranging from ∼5 to 60% of total circulating CD8<sup>+</sup> T cells (Van Braeckel-Budimir and Harty, 2014). Similarly, adoptive transfer of ∼10<sup>7</sup> activated specific CD8<sup>+</sup> T cells, which totalize ∼26 to 60% of CD8<sup>+</sup> T cells circulating in the blood, is required to sterilize the infection in the liver, while the transfer of 10<sup>6</sup> CD8<sup>+</sup> T cells, which represents ∼3% of CD8<sup>+</sup> T cells circulating in the blood, is not enough to completely protect mice against sporozoite challenge (Kimura et al., 2013). In addition, interactions between CD8<sup>+</sup> T cells and infected hepatocytes could not be observed after adoptive transfer of 10<sup>6</sup> primed CD8<sup>+</sup> T cells to infected mice (Cabrera et al., 2013), suggesting that the recognition of infected hepatocytes is dependent on high numbers of specific CD8<sup>+</sup> T cells. In humans, however, the levels of CD8<sup>+</sup> T cells correlated with protection seem to be much lower than in rodents (Ewer et al., 2013). Protective activity is dependent not only on the quantity, but also on the quality of CD8<sup>+</sup> T cells. For example, high expression levels of cell adhesion molecules such as CD44 and VLA-4 are correlated with the *in vivo* anti-parasite activity of CD8<sup>+</sup> T cells, but not with their *in vitro* cytotoxic activity (Rodrigues et al., 1992). These molecules have been implicated in homotypic and heterotypic adhesion of lymphocytes, in the trafficking of T cells to inflamed site (Nandi et al., 2004), and could play an important role in the clustering of T cells at the site of infection (next sections).

Very little is known about the nature of protective parasite epitopes presented on the surface of infected hepatocytes by class I major histocompatibility complex molecules (MHC I). In rodent models, protective MHC I-restricted epitopes were described in the circumsporozoite protein (CSP, Romero et al., 1989; Weiss et al., 1992; Rodrigues et al., 1994; Schmidt et al., 2008) and in the thrombospondin-related anonymous protein (TRAP, Hafalla et al., 2013). Both CSP and TRAP are membrane proteins expressed mainly in sporozoites. In humans, immunization using adjuvanted, truncated CSP induced only partial protection without a detectable specific CD8<sup>+</sup> T cell response (Moorthy and Ballou, 2009). Immunization using TRAP delivered by viral vectors, which elicit robust T-cell responses, induced also a partial protection which was correlated with the frequency of monofunctional interferon gamma (IFNγ)-producing CD8<sup>+</sup> T cells (Ewer et al., 2013). Intriguingly, several attempts to identify new epitopes targeted by CD8<sup>+</sup> T cells among thousands of profiled peptides, mini-genes or genes covering hundreds of parasite proteins, have revealed only a few new PE candidate antigens, but none of them induced protection against sporozoite infection (Mishra et al., 2011; Murphy et al., 2013; Hafalla et al., 2013). Whether the difficulty to unravel new protective epitopes is a consequence of the method of screening, which relies on the secretion of IFNγ by CD8<sup>+</sup> T cells detected by ELISPOT, or of immunization, which may not reach the critical CD8<sup>+</sup> T cell threshold necessary for protection, remains to be determined.

Given the scarcity of protective CD8 epitopes, another convenient way to study CD8<sup>+</sup> T cell responses against infected hepatocytes is to use the epitope of ovalbumin (OVA) fused to endogenous and exogenous antigens expressed in malaria PE stages (Cockburn et al., 2011; Kimura et al., 2013; Montagna et al., 2014). This strategy showed that membrane, cytoplasmic and tubulovesicular network exported proteins can harbor epitopes that are presented on the surface of infected hepatocytes, leading to the parasite elimination by MHC I–restricted (H-2K<sup>b</sup> ), OVA-specific CD8<sup>+</sup> T cells (OT-I cells). Although antigen-presentation is dependent on the transporter associated with antigen processing (TAP; Cockburn et al., 2011; Kimura et al., 2013), which translocates peptides from the cytosol into the endoplasmic reticulum where they are loaded onto MHC I molecules, the mechanism by which these membrane, cytoplasmic and exported parasite antigens cross the parasitophorous vacuole membrane and reach the host cell cytoplasm is still controversial and unclear (Singh et al., 2007; Cockburn et al., 2011; Montagna et al., 2014).

The mechanisms by which CD8<sup>+</sup> T cells eliminate liver stages are also puzzling. Systemic depletion of IFNγ consistently abolishes the sterile protection induced by irradiated sporozoites in rodent models (Schofield et al., 1987; Doolan and Hoffman, 2000). However, activated IFNγ <sup>−</sup>/<sup>−</sup> CD8<sup>+</sup> T cells harboring transgenic T-cell receptors (TCRs), which can recognize specific cognate epitopes presented on the surface of infected hepatocytes, are still capable of controlling infection as their IFNγ-proficient counterparts (Chakravarty et al., 2008; Kimura et al., 2013). The same is observed for FasL and/or perforin knockout CD8<sup>+</sup> T cells (Morrot and Zavala, 2004; Kimura et al., 2013). Finally, sterile protection is only lost in a proportion of animals injected with double IFNγ and perforin knockout CD8<sup>+</sup> T cells (Kimura et al., 2013). Altogether these loss-of-function studies depict a scenario where the elimination of liver stages by specific CD8<sup>+</sup> T cells seems to be a complex process involving several and redundant effector molecules and probably other IFNγ-producing immune cells.

#### **CD8**<sup>+</sup> **T CELL–DEPENDENT INFLAMMATORY FOCI AND THE DEATH OF LIVER STAGES**

While molecular mechanisms underlying the elimination of liver stages by CD8<sup>+</sup> T cells are still unclear, the cellular events leading to the death of these hepatic parasites are only slowly being revealed. In 1989, multiple inflammatory foci containing numerous CD11b+, CD8+, and at lesser extent CD4<sup>+</sup> cells were first observed in the liver of BALB/c mice immunized with *P. berghei* (Pb) irradiated sporozoites, 43 h after challenge with normal sporozoites. Although no parasites could be observed in association with these infiltrates, the formation of these cellular foci was dependent on the presence of CD8<sup>+</sup> T cells (Hoffman et al., 1989).

Several years later, DNA *in situ* hybridization revealed remnants of *Pb* parasites in close association with cellular infiltrates in the liver of rats immunized with irradiated sporozoites and challenged with normal sporozoites (Scheller et al., 1997). The number of infiltrates increased after 24 h post-challenge and coincided with the decrease in the number of liver stages in the immunized rats. CD4<sup>+</sup> and CD8<sup>+</sup> T cells were also identified in these infiltrates. The number of CD4<sup>+</sup> cells was maintained constant at 31 h and 44 h post-infection, while the number of CD8<sup>+</sup> T cells was four and sixfold superior to those of CD4<sup>+</sup> T cells at these respective time points.

Notably, systemic inhibition of nitric oxide (NO) production by aminoguanidine reduced the number of inflammatory foci after 24 h post-infection with a clear diminution of the number of CD8<sup>+</sup> lymphocytes in the focal infiltrates (Scheller et al., 1997). This treatment also abolished the elimination of liver stages in the immunized animals indicating that the production of NO is critical for the accumulation of CD8<sup>+</sup> T cells, which Bayarsaikhan et al. Cooperative killing of liver stages

are necessary for the parasite elimination. However, the source of inducible NO synthase in the liver of immunized rats challenged with sporozoites seems to be restricted to infected hepatocytes (Klotz et al., 1995). Since NO produced by infected hepatocytes is involved in the elimination of parasites *in vitro* in the absence of T cells (Nussler et al., 1993; Mellouk et al., 1994), it is not possible to distinguish if aminoguanidine abolished elimination of liver stages in the immunized animal by the direct inhibition of NO anti-plasmodial activity, by the inhibition of inflammatory responses that induce the accumulation of CD8<sup>+</sup> T cells around the infected hepatocytes, or both. Studies using specific CD8<sup>+</sup> T lymphocytes or liver cells that are deficient in the inducible synthesis of NO could precise the role of this molecule in the killing of liver stages.

#### **ANTIGEN-DRIVEN INFLAMMATORY FOCI**

Recently, *in vivo* imaging revealed CD8<sup>+</sup> T cell infiltrates in close association with infected hepatocytes in BALB/c mice immunized with irradiated *P. yoelii* (Py) sporozoites (Cockburn et al., 2013). The use of fluorescent CD8<sup>+</sup> T cells harboring transgenic TCRs, specific for epitopes expressed by the parasite and presented by MHC I molecules on the surface of infected hepatocytes, also showed that cluster formation mediated by CD8<sup>+</sup> T cells is antigen specific. Activated CD8<sup>+</sup> T cells, which recognize specifically a *Py*CSP-epitope, clustered around and eliminated *Py* infected hepatocytes. Conversely activated OT-I cells did not cluster or eliminate *Py* and *Pb* infected hepatocytes, but readily clustered around and eliminated hepatocytes infected with *Pb* expressing the CD8 epitope of OVA (Cockburn et al., 2013; Kimura et al., 2013). The frequency distribution of *Py*CSP-specific CD8<sup>+</sup> T cells around infected hepatocytes fitted a mathematical model where the recruitment of T cells was density-dependent, indicating that activated CD8<sup>+</sup> T cells were attracted to a cluster. The close apposition of CD8<sup>+</sup> T cells with infected hepatocytes suggests that this first step of specific recognition and recruitment is initiated by the direct contact between these two cells (**Figure 1A**). Hepatocytes traversed by sporozoites are also thought to present antigens to CD8<sup>+</sup> T cells. Although traversed hepatocytes do not alter the elimination of infected cells by CD8<sup>+</sup> T cells *in vitro* (Bongfen et al., 2007), the role of liver cells traversed by sporozoites (Mota et al., 2001; Tavares et al., 2013a,b) located adjacent to infected hepatocytes cannot be formally excluded in the process of initiation and formation of these cellular inflammatory foci (**Figure 1B**). This capacity of CD8<sup>+</sup> T cells to recognize a cognate antigen and recruit other CD8<sup>+</sup> T cells to the site of infection seems to be a general and novel function of CD8<sup>+</sup> T cells, as reported recently in a model of mouse reproductive tract infection by the lymphocytic choriomeningitis virus (Schenkel et al., 2013).

Surprisingly, when activated *Py*CSP-specific and non-specific CD8<sup>+</sup> T cells were transferred together in mice infected with *Py* sporozoites, non-specific CD8<sup>+</sup> T cells, which usually did not cluster around *Py* infected hepatocytes, modified their behavior and were recruited together with *Py*CSP-specific CD8<sup>+</sup> T cells to the site of infection (Cockburn et al., 2013). In a tumor model, activated OT-I cells were also responsible for the deep infiltration and accumulation of activated non-specific T cells in OVA-expressing EL4 tumors (Boissonnas et al., 2007), indicating

**FIGURE 1 | Model of the elimination of malaria liver stage by a CD8**<sup>+</sup> **T cell-dependent inflammatory cell cluster. (A)** Parasite antigens cross the parasitophorous vacuole membrane (PVM), are processed and presented on the surface of the infected hepatocyte by MHC I molecules, and/or **(B)** are released in the cytoplasm and presented on the surface of hepatic cells (hepatocytes, Kupffer cells, endothelial cells—figuratively represented by the pink cells) traversed by sporozoites. **(A,B)** Parasite-specific, activated CD8<sup>+</sup> T cell (red cell) recognizes its cognate antigen and **(C)** recruits other specific and non-specific (purple cells) CD8<sup>+</sup> T cells, and presumably other leukocytes (orange cells) to the site of infection. **(D)** These immune cells cluster around the infected hepatocyte, leading to the elimination of the malaria liver stage and presumably of the infected hepatocyte. For clarity and due to the lack of information about its position during cluster formation, the sinusoidal barrier is not represented in the model.

that CD8<sup>+</sup> T cells not only exert a direct cytotoxic activity on target cells, but can also orchestrate a focal inflammatory response by the recognition of a specific antigen and further recruitment of specific and non-specific T cells to the site of infection or tumor growth. This inflammatory role is in agreement with the recent observation that CD8<sup>+</sup> T cells express a burst of inflammatory cytokines immediately after cognate antigen stimulation recruiting diverse cellular types involved in inflammatory reactions (Sung et al., 2013).

#### **MULTIPLICITY OF PARASITE DEATH PHENOTYPES**

Dynamic imaging revealed how clusters of activated CD8<sup>+</sup> T cells eliminate liver stages *in vivo* (Cockburn et al., 2013; Kimura et al., 2013). The utilization of a vitality index (VI) based on the fluorescence intensity of GFP-expressing liver stages permitted the monitoring of parasite viability/death over hours of observation after the transfer of activated and specific CD8<sup>+</sup> T cells. Strikingly, killing of liver stages was a lasting process, which on average occurred after several hours of association between CD8<sup>+</sup> T cells and infected hepatocytes. In addition, the kinetics of the VI of fluorescent parasites allowed the discrimination of at least three distinct death phenotypes (DPs) associated with the presence of activated CD8<sup>+</sup> T cells. An abrupt decrease in the parasite VI followed by the diffusion of a weak GFP signal through the host cell cytoplasm characterized the first DP. The second DP was characterized by a slow decrease in the VI over hours of interaction. The blebbing of the infected hepatocyte with the release of parasite material into the sinusoids characterized the third DP. All these distinct DPs suggest again that parasite elimination could rely on multiple and redundant mechanisms of killing.

A better understanding of these killing mechanisms might be achieved using functional *in vivo* imaging. This strategy is a double approach that combines the quantitative imaging of mutant cells with the imaging of non-mutant cells using specific fluorescent reporters to investigate function (Tavares et al., 2013a). For example, the correlation between the ablation of a cytotoxic effector molecule in protective CD8<sup>+</sup> T cells and the loss of a given DP could indicate a causal relation between these two variables. Complementary, the use of fluorescent markers that report the level of expression of cytotoxic molecules (Croxford and Buch, 2011; Mouchacca et al., 2013) could be used to discriminate different populations of protective CD8<sup>+</sup> T cells and their relationship with the DPs could be assessed by *in vivo* imaging. The latter approach could also reveal what is the activation phenotype of CD8<sup>+</sup> T cells associated with efficient homing to the liver, recognition of infected hepatocytes, clustering formation and killing of liver stages.

#### **CLUSTERING OF CELLS FACILITATES ELIMINATION OF LIVER STAGES**

Qualitatively a few CD8<sup>+</sup> T cells are sufficient to rapidly eliminate a liver stage *in vitro* (Trimnell et al., 2009), however, the killing process *in vivo* seems to be much longer and complex, involving multiple cells (Cockburn et al., 2013). Whether these differences are due to intrinsic characteristics of CD8<sup>+</sup> T cells used in these studies or are a consequence of limitations imposed by an *in vitro* system is not yet determined. The liver possesses an immune-privileged environment, which confers a relatively high resistance against CD8<sup>+</sup> T cell responses (Pircher et al., 2006). This fact could explain the long time that these cells require to eliminate liver stages *in vivo*. Cell clustering triggered by antigenspecific CD8<sup>+</sup> T cells might possibly facilitate the process of parasite killing by augmenting the local concentration of protective cellular and molecular effectors. To test this hypothesis, cluster formation was inhibited by targeting chemokine signaling. Chemokines act on T cells via G-Protein Coupled Receptors (GPCRs) signaling. Pertussis toxin (PTx) was used to inhibit GPCR signaling through the ADP-ribosylation and uncoupling of G-Proteins (Cotton and Claing, 2009).  Although this pharmacological approach indicates that the clustering of cells triggered by *Py*CSP-specific CD8<sup>+</sup> T cells around infected hepatocytes facilitates the elimination of malaria liver stages, the identification of the chemokine(s) involved in the recruitment of cells to a cluster is still missing. The discovery of these molecules will permit a precise assessment, e.g., using knockout animals, of the role of cell clustering in the elimination of malaria liver stages.

#### **CONCLUDING REMARKS AND PERSPECTIVES**

Altogether these data suggest that CD8<sup>+</sup> T cell-dependent elimination of liver stages is a complex and cooperative process involving multiple cells and effector molecules. An elevated number of CD8<sup>+</sup> T cells with a proper binding phenotype seem to be required for finding and eliminating the scarce population of infected hepatocytes in the liver. Following antigen-specific recognition of the site of infection by activated CD8<sup>+</sup> T cells, both specific and non-specific CD8<sup>+</sup> T cells and likely other immune cells are recruited to the site of infection. Finally infected hepatocytes are cleared by these antigen-driven inflammatory foci, displaying a multiplicity of DPs. This mechanism of killing places CD8<sup>+</sup> T cells as an essential linker between a specific adaptive immune response, which starts with the recognition of an infected cell, and a non-specific immune response, characterized by the recruitment of inflammatory cells to the site of infection and ensuing elimination of liver stages (**Figure 1**). This multi-cellular cooperative model of killing can also incorporate the relative importance of other cellular types, like CD4<sup>+</sup> T cells and other IFNγ-producing cells, in the foci composition and protection. It may also explain the general dependence on IFNγ for sterile immunity but the dispensable role of this cytokine for protection induced by parasite-specific IFNγ −/− CD8<sup>+</sup> T cells.

Important topics related to the formation, composition and killing efficacy of these CD8<sup>+</sup> T cell-dependent inflammatory foci are still unclear and need further investigation. One of these topics is the identification of novel protective CD8 epitopes presented on the surface of hepatocytes (Crabb et al., 2012) and the verification of their concerted action with other known protective epitopes, such as those described in CSP and TRAP. This approach could reveal a possible synergistic, additive, neutral or antagonistic effect on cluster formation and protection. Another important subject to address is the contribution of CD8<sup>+</sup> T cells, which cannot recognize infected hepatocytes, but are specifically activated in the lymph node that drains the bite site, for example, by mosquito saliva or sporozoite antigens (Chakravarty et al., 2007), in foci formation and elimination of liver stages. Once it is determined that the recruitment of these activated CD8<sup>+</sup> T cells to infected hepatocytes can improve the killing of liver stages, a vaccination strategy using protective epitopes, presented by infected hepatocytes, and inflammatory epitopes, which might improve killing by focal inflammation, could be envisaged and tested. Similarly the role of non-specific innate immune cells in cluster formation and killing activity is also an open question. It is well documented that inflammatory infiltrates are associated with the natural resistance of naïve rodents to hepatic infection (Khan and Vanderberg, 1991), but their role in immunized animals is not known. In addition, depletion studies using multiple doses of anti-asialo GM1 antibodies partially abrogated protection induced by irradiated sporozoites in several mouse strains (Doolan and Hoffman, 2000). This treatment is known to efficiently deplete NK cells and basophils, and despite the expression of asialo GM1 on subpopulations of NKT, CD8<sup>+</sup> T and γδ T cells, no significant depletion was observed after a single dose of anti-asialo GM1 antibodies in C57BL/6 mice (Nishikado et al., 2011). This suggests that NK cells, basophils and other innate immune cells could also participate in the focal inflammation orchestrated by specific CD8<sup>+</sup> T cells. Finally the unraveling of the molecular determinants of highly protective CD8<sup>+</sup> T cells is crucial for defining the type of effector cells a vaccine should generate. All this information could be very useful for the design of an immunization strategy aiming at the efficient elimination of malaria liver stages mediated by CD8<sup>+</sup> T cells.

#### **ACKNOWLEDGMENTS**

We would like to thank our collaborators, Institut Pasteur and French National Research Agency—ANR PlasmoPEP (RA), Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25113717 (KY) and Program for Nurturing Global Leaders in Tropical and Emerging Communicable diseases of Nagasaki University (GB) for the support, and Robert Menard for critically reading the manuscript. We also apologize for all colleagues whose work was not cited here due to space limitations or unawareness.

#### **REFERENCES**

T cell elimination of malaria from hepatocytes. *Science* 244, 1078–1081. doi: 10.1126/science.2524877

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 October 2014; accepted: 14 January 2015; published online: 04 February 2015.*

*Citation: Bayarsaikhan G, Akbari M, Yui K and Amino R (2015) Antigen-driven focal inflammatory death of malaria liver stages. Front. Microbiol. 6:47. doi: 10.3389/fmicb.2015.00047*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology.*

*Copyright* © *2015 Bayarsaikhan, Akbari, Yui and Amino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### A sufficient role of MHC class I molecules on hepatocytes in anti-plasmodial activity of CD8**+**T cells in vivo

#### *Jing Huang1\*, Tiffany Tsao1, Min Zhang2 , Urvashi Rai 1 †, Moriya Tsuji <sup>1</sup> and Xiangming Li <sup>1</sup>*

<sup>1</sup> HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center – The Rockefeller University, New York, NY, USA <sup>2</sup> Department of Pathology, New York University, New York, NY, USA

#### *Edited by:*

Urszula Krzych, Walter Reed Army Institute of Research, USA

#### *Reviewed by:*

Alexander Pichugin, Walter Reed Army Institute of Research, USA Ian Andrew Cockburn, Australian National University, Australia Jelena Vladimirovna Levitskaya, Johns Hopkins Bloomberg School of Public Health, USA

#### *\*Correspondence:*

Jing Huang, HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center – The Rockefeller University, New York, NY 10016, USA e-mail<EMAIL_ADDRESS>
#### *†Present address:*

Urvashi Rai, Biomedical Sciences Training Program, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA Although CD8+ T cells are shown to mediate the protective immunity against the liver stages of malaria parasites in mice, whether the direct presentation of malaria antigen by major histocompatibility complex (MHC) class I molecules expressed on the liver of infected host is required for anti-plasmodial activity of CD8+ T cells is still unknown. Presently, there is only one CD8+ epitope, SYVPSAEQI, derived from the circumsporozoite protein of Plasmodium yoelii (PyCS), that mediates anti-malarial protection and is presented in the context of a K<sup>d</sup> molecule. Therefore, to investigate the mode of anti-plasmodial activity of CD8<sup>+</sup> T cells, we have previously generated C57BL/6 transgenic (Tg) mice, in which a K<sup>d</sup> molecule is expressed only on hepatocyte (Alb-Kd) or dendritic cell (DC; CD11c-Kd), by using albumin promoter or CD11c promoter, respectively. We have also generated MHC-I-K<sup>d</sup> Tg mice, which express the K<sup>d</sup> molecule under the MHC class I (MHC-I) promoter, as a positive control. From splenocytes collected from CD11c-Kd Tg mice immunized with a synthetic peptide, SYVPSAEQI, which corresponds to the CD8+ T-cell epitope of PyCS, emulsified in incomplete Freund's adjuvant , a PyCS-specific CD8+ T-cell line was generated. This PyCSspecific CD8+T-cell line was then adoptively transferred into a cohort of either MHC-K<sup>d</sup> Tg or Alb-K<sup>d</sup> Tg mice listed above, as well as wild-type C57BL/6 mice. Then both transferred and non-transferred mice were challenged with live malaria parasites. We found that the adoptive transfer of a PyCS-specific CD8+ T-cell line resulted in a significant inhibition of the parasite burden in the liver of Alb-K<sup>d</sup> Tg, as well as MHC-I-K<sup>d</sup> Tg mice, but not of C57BL/6 mice. These results indicate that the K<sup>d</sup> molecule expressed by hepatocytes is sufficient in mediating the anti-plasmodial activity of PyCS-specific CD8+ T cells in vivo.

#### **Keywords: malaria, CD8+ T cell, liver, MHC class I, transgenic mouse**

#### **INTRODUCTION**

Malaria is a severe disease that ranks among the most prevalent infections in tropical areas throughout the world. Approximately 250–300 million people become infected yearly with relatively high rates of morbidity and mortality. The WHO estimates that every year nearly one million children die of malaria in Africa alone (World Health Organization [WHO], 2008). The widespread occurrence and the increasing incidence of malaria in many countries, caused by drug resistant parasites and insecticide resistant vectors (*Anopheles mosquitoes*), underscore the need for developing new methods of controlling this disease, which includes more effective vaccines.

A number of mouse studies to date using *Plasmodium yoelii* and *P. berghei* parasites for challenge have shown that protective immunity against pre-erythrocytic stages is mediated in part by T cells, particularly CD8+ T cells (Weiss et al., 1988, 1992; Rodrigues et al., 1991, 1997; Tsuji et al., 1998; Sano et al., 2001; Schmidt et al., 2008). Then, the essential role of major histocompatibility complex class I (MHC-I) molecules in mediating CD8+ T-celldependent anti-malarial immunity was shown by the study in which adoptive transfer of malaria-immune splenocytes into β2 microglobulin (β2 m)-deficient mice failed to confer protection (White et al., 1996). Using transgenic (Tg) mice that express a Tcell receptor (TCR), based on the TCR sequence of CD8+ T cells that recognize an immunodominant T-cell epitope of the PyCS protein, SYVPSAEQI, a recent study showed that the Tg T cells do not require bone marrow-derived antigen-presenting cells (APCs) for protection upon adoptive transfer; instead, they recognize antigen on parenchymal cells, presumably parasitized hepatocytes (Chakravarty et al., 2007). These studies all together strongly suggest that MHC-I molecules expressed by malaria-infected hepatocytes play a key role in mediating the anti-plasmodial activity of CD8+ T cells *in vivo*. However, it is still unknown to which extent MHC-I molecules expressed by hepatocytes mediate the anti-plasmodial activity of CD8+ T cells *in vivo*.

The immunodominant T-cell epitope, SYVPSAEQI, described above is presented by H-2Kd (Kd) molecules to CD8<sup>+</sup> T cells and is known to be the only epitope that can induce protective CD8+ T cells against malaria. Therefore, to provide answers concerning the mechanisms underlying the anti-plasmodial activity of CD8+ T cells, we have taken an approach in which Tg C57BL/6 mice, expressing H-2K<sup>d</sup> molecules only on the surface of hepatocytes (Alb-Kd), DCs (CD11c-Kd), or all nucleated cells (MHC-I-Kd), have been generated (Huang et al., 2013) and used in the current studies.

#### **MATERIALS AND METHODS**

#### **PARASITES AND ANIMALS**

Female *Anopheles stephensi* mosquitoes infected with *P. yoelii* 17XNL strain were purchased from the New York University insectary. *P. yoelii* sporozoites were isolated from the salivary glands of infected *A. stephensi* mosquitoes 14 days after the mosquitoes had received an infectious blood meal (Huang et al., 2014). Six- to eight-week-old C57BL6 mice were purchased from Taconic (Germantown, NY, USA). Three tissues specific H-2Kd Tg mice models, Alb-Kd, CD11c-Kd, and major histocompatibility complex-I-K<sup>d</sup> (MHC-I-Kd) Tg mice models, were established in our lab, in which H-2Kd were expressed under the control of albumin, CD11c, and MHC I promoters, respectively, in C57BL/6 mice (Huang et al., 2013, 2014). All mice were maintained under standard conditions in The Laboratory Animal Research Center of The Rockefeller University. Furthermore, all animal experiments were carried out in strict accordance with the Policy on Humane Care and Use of Laboratory Animals of the United States Public Health Service. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at The Rockefeller University (Assurance # A3081-01).

#### **PEPTIDE, TETRAMER, AND CULTURE MEDIUM**

A peptide, SYVPSAEQI, which corresponds to the CD8+ T cell epitope of the CS protein of *P. yoelii* (PyCS), was synthesized by Peptide 2.0 Inc. (Chantilly, VA, USA). An allophycocyanin (APC) labeled H-2Kd/SYVPSAEQI-tetramer was generated and provided to us by the NIH Tetramer Core Facility at Emory University. As culture medium we used DMEM-high glucose (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Thermo Scientific, Waltham, MA, USA) and 1% supernatant derived from phorbol myristate acetate (Fisher Scientific, Pittsburgh, PA, USA) activated EL-4 cells (Huang et al., 2013).

#### **GENERATION OF A PyCS-SPECIFIC CD8<sup>+</sup> T-CELL LINE**

CD11c-K<sup>d</sup> Tg mice were immunized twice with a 2-week interval and intra-peritoneally with10 μg of the synthetic peptide, SYVPSAEQI, emulsified in incomplete Freund's adjuvant (IFA; Sigma-Aldrich, St. Louis, MO, USA). Ten days after the last immunization, splenocytes collected from immunized CD11c-Kd mice were stimulated with radiation-attenuated (3000-rad) splenocytes isolatedfrom naiveMHC-I-K<sup>d</sup> mice pulsed with SYVPSAEQI peptide. We repeated the stimulation for a few times every 10 days, as described Rodrigues et al. (1991), and 10 days after the last stimulation, the frequency of PyCS-specific CD8+ T cells among the T-cell line was determined by either Tetramer staining or an ELISpot assay.

#### **TETRAMER STAINING AND ELISPOT ASSAY**

The specificity and frequency of a PyCS-specific CD8+ T-cell line was determined by a flow cytometric analysis after staining the T-cell line with antibodies against various CD molecules and an APC-labeled H-2Kd/SYVPSAEQI-tetramer. All the antibodies used in this study were purchased from BioLegend (San Diego, CA, USA). Briefly, cells were first incubated with unlabeled anti-CD16/CD32 antibody (Clone 16) for 10 min at 4◦C. Then the cells were incubated with FITC-labeled anti CD3 antibody (17A2), Pacific Blue-labeled anti-CD8 alpha antibody (clone 53–6.7), and APC-labeled H-2Kd/SYVPSAEQI-tetramer at room temperature for 30 min. A flow cytometric analysis was performed using an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). The frequency of SYVPSAEQI-specific, IFN-γ– secreting T cells was determined by an ELISpot assay, as previously described Huang et al. (2013, 2014). Briefly, 1000 cells of the T cell line were co-cultured with 5 <sup>×</sup> <sup>10</sup><sup>5</sup> EL-4-K<sup>d</sup> cells (Huang et al., 2013) loaded with SYVPSAEQI in a well of an ELISpot plate. Twenty-four hours later, the relative number of IFN-γ secreting cells was determined by counting the number of spots that corresponds to IFN-γ-secreting cells using a stereomicroscope. The number of IFN-γ secreting cells after co-culturing with EL-4-Kd cells without SYVPSAEQI peptide was used as negative control.

#### **ADOPTIVE TRANSFER AND PARASITE CHALLENGE**

Ten million cells of PyCS-specific CD8+ T-cell line were adoptively transferred intravenously (i.v.) into each mouse (Rodrigues et al., 1991). Twenty-four hours later, 1 <sup>×</sup> 104 viable *P. yoelii* sporozoites were inoculated to each transferred, as well as non-transferred mice by tail vein injection (Rodrigues et al., 1991).

#### **ASSESSMENT OF PARASITE BURDEN IN THE LIVER**

Parasite burden in the liver was determined 42 h after sporozoite challenge by measuring the amount of parasite-specific 18S rRNA in the liver of challenged mice, using a real-time quantitative RT-PCR with the 7500 Fast Real-Time PCR System (Life Technologies, Grand Island, NY, USA; Huang et al., 2014). Parasite burden was described as a ratio of the absolute copy number of parasite-specific 18S rRNA to that of mouse GAPDH.

#### **STATISTICAL ANALYSES**

Statistical analyses were done using GraphPad Prism (version 5.03; GraphPad Software Inc., La Jolla, CA, USA). All data were expressed as the mean ± SD of three mice. Statistical analyses of experimental and control data were evaluated by one-way ANOVA and Student's *t-*test. A value of *p*<0.05 was considered statistically significant.

#### **RESULTS**

#### **FREQUENCY OF PyCS-SPECIFIC CD8<sup>+</sup> T CELLS EXPANDED** *IN VITRO* **FROM ISOLATED SPLENOCYTES OF CD11c-Kd MICE IMMUNIZED WITH SYVPSAEQI PEPTIDE**

CD11c-K<sup>d</sup> Tg mice were immunized twice with SYVPSAEQI peptide emulsified in IFA with a 2-week interval. Then splenocytes from peptide-immunized CD11c-K<sup>d</sup> Tg mice were collected for the expansion of a PyCS-specific CD8+ T-cell line *in vitro*, which was achieved by stimulating the cells for a few times with irradiated MHC-I-Kd Tg mouse-derived splenocytes pulsed with the SYVP-SAEQI peptide in a 10–14-day interval, as similarly performed previously (Rodrigues et al., 1991). After successful expansion of a PyCS-specific CD8+ T-cell line, the specificity andfrequency of the PyCS-specific CD8+ T-cell line were determined by either APClabeled H-2Kd/SYVPSAEQI-tetramer staining or IFN-γ ELISpot assay, as shown in **Figure 1**. The tetramer staining results depicted

**by either a tetramer staining or an ELISpot assay.** Frequency of PyCS-specific CD8+ T cells among the T-cell line was determined by either a tetramer staining **(A)** or an IFN-γELISpot assay **(B)**. In **(A)**, CD3+ T cells gated with anti-CD8 antibody were stained with APC-labeled H-2Kd/SYVPSAEQI-tetramer. The number shows the percentage of CD8<sup>+</sup>

T cells that are positive with H-2Kd/SYVPSAEQI-tetramer. In **(B)**, 1000 cells of the T-cell line were co-cultured with 5 <sup>×</sup> <sup>10</sup><sup>5</sup> EL-4-K<sup>d</sup> cells loaded with SYVPSAEQI in a well of an ELISpot plate, and 24 h later, the relative number of IFN-γ-secreting cells was determined by an ELISpot assay. The number of IFN-γ-secreting cells after co-culturing with EL-4-K<sup>d</sup> cells without SYVPSAEQI peptide loading was used as a negative control.

that more than two third of the cells of a PyCS-specific CD8+ Tcell line could be identified as H-2Kd/SYVPSAEQI tetramer CD8<sup>+</sup> T cells (**Figure 1A**). By assessing the ELISpot assay, more than 700 cells out of 1,000 cells of the T cell line were found to get activated by the SYVPSAEQI peptide and secrete IFN-γ, whereas in the absence of the SYVPSAEQI peptide, the cells did not display any responses, indicating that there was no non-specific or autoreactive T cells present in the T cell line (**Figure 1B**). This indicates

that a majority (>65–70%) of the population in the T cell line are specific to the SYVPSAEQI epitope.

#### **INHIBITION OF** *P. yoelii* **HEPATIC STAGE DEVELOPMENT IN MHC-Kd MICE AND Alb-Kd MICE AFTER ADOPTIVELY TRANSFERRING PyCS-SPECIFIC CD8<sup>+</sup> T CELLS**

Now that we generated a PyCS-specific CD8+ T-cell line having more than two third of the cells specific to SYVPSAEQI peptide,

**PyCS-specific CD8+ T cells, as determined by a real-time qRT-PCR.** Each mouse of MHC-K<sup>d</sup> Tg mice, Alb-K<sup>d</sup> Tg mice, and wild-type C57BL/6J mice (three mice per group) received 1 <sup>×</sup> <sup>10</sup>7cells of a PyCS-specific CD8+ T-cell line intravenously. Twenty-four hours later, the transferred, as well as non-transferred Tg mice and wild-type C57BL/6 mice, number of parasite-specific 18S rRNA to that of mouse GAPDH using a real-time qRT-PCR. The mice that do not receive PyCS-specific CD8+ T-cell transfer served as negative controls. Error bars represent means ± SEM (n = 3). A value of p < 0.05 was considered statistically significant, whereas N.S. means "not significant."

we sought to address one of the key questions regarding the manner in which CD8+ T cells recognize and eliminate the hepatic stage of malaria *in vivo*. More specifically, we aimed to determine the role of MHC-I molecules expressed on hepatocytes in mediating CD8+ T cells' recognition of malaria-infected hepatocytes and their anti-plasmodial activity *in vivo*. For this purpose, we adoptively transferred 1 <sup>×</sup> <sup>10</sup><sup>7</sup> cells of PyCS-specific CD8<sup>+</sup> Tcell line to three groups (three mice each): MHC-I-Kd Tg mice (as a positive control), Alb-K<sup>d</sup> Tg mice and wild-type C57BL/6 mice (as a negative control). Respective Tg mice that do not receive the PyCS-specific CD8+ T-cell transfer were used as a negative control for each group of transferred Tg mice. All experimental mice were then challenged with 1 <sup>×</sup> 104 live *P. yoelii* sporozoites. Forty-two hours after the challenge, the livers were collected from challenged mice, and the liver parasite burden was determined by measuring the parasite-specific rRNA by real-time PCR and quantified by a ratio of the absolute copy number of parasite-specific 18S rRNA to that of mouse GAPDH. After the *P. yoelii* sporozoites challenge, the PyCS-specific CD8+ T-cell line inhibited almost 50% of the parasite burden in the liver of MHC-I-K<sup>d</sup> Tg mice, but not in C57BL/6 mice (**Figure 2**). Most importantly, a PyCS-specific CD8+ T-cell line transferred to Alb-Kd Tg mice could inhibit (55%) the liver stage development as potently as those transferred to MHC-I-K<sup>d</sup> Tg mice. These results indicate that K<sup>d</sup> molecules expressed on hepatocytes are sufficient in mediating the anti-parasitic effect of PyCS-specific CD8+ T cells *in vivo*.

#### **DISCUSSION**

The role of MHC-I molecules in mediating CD8+ T celldependent immunity against the liver stages of rodent malaria parasites has been shown by a few studies as described in the introduction section (White et al., 1996; Chakravarty et al., 2007). These studies have clearly led to a conclusion that CD8+ T cells are unable to confer protection in the absence of MHC-I molecules (White et al., 1996) and that CD8+ T cells do not require bone marrowderived APCs for protection (Chakravarty et al., 2007). However, whether CD8+ T cells need to recognize MHC-I expressed on hepatocytes in order to exert anti-plasmodial effector activity is yet unknown. Therefore, for the purpose of addressing this key question, we have generated B6 Tg mice that express Kd molecule only on hepatocyte (Alb-Kd; Huang et al., 2013) in addition to those that express K<sup>d</sup> molecules in all nucleated cells (MHC-I-Kd; Huang et al., 2013) and performed a set of adoptive transfer experiments, in which malaria-specific CD8+ T cell-line was transferred to Alb-Kd Tg mice, MHC-I-K<sup>d</sup> Tg mice (as a positive control), and B6 mice (as a negative control).

We first sought to establish a PyCS-specific CD8+ T-cell line from MHC-K<sup>d</sup> Tg mice, which is a rather straightforward approach. However, such T cell-line bears K<sup>d</sup> molecule, which should cause allogeneic reaction to wild-type C57BL/6 mice (K<sup>d</sup> negative) upon being adoptively transferred. Therefore, we decided to generate a PyCS-specific CD8+ T-cell line from CD11c-Kd Tg mice, in which DCs express K<sup>d</sup> molecules, and can present SYVPSAEQI peptide to CD8<sup>+</sup> T cells that lack K<sup>d</sup> expression.

In the present study, we found that a PyCS-specific CD8+ T-cell line adoptively transferred to Alb-Kd Tg mice inhibited more than 50% of the liver stage development, which is equal to the degree of inhibition by the CD8<sup>+</sup> T cell-line found in MHC-I-K<sup>d</sup> Tg mice. These results indicate that K<sup>d</sup> molecules expressed on hepatocytes play a major role in mediating the effector phase of anti-malaria CD8+ T-cell response *in vivo*.

As we previously shown, we have also generated C57BL/6 Tg mice that express K<sup>d</sup> only on DCs (CD11c-K<sup>d</sup> Tg) and macrophages (hCD68-Kd Tg; Huang et al., 2013) for the purpose of determining the role of cells other than hepatocytes, like Kupffer cells and DCs, in triggering PyCS-specific CD8+ T cells. However, the difficulty of expanding and generating a large number of PyCS-specific CD8<sup>+</sup> T cells from CD11c-K<sup>d</sup> Tg or hCD68-K<sup>d</sup> Tg mice, thus far, has hampered us from determining the role of DCs and Kupffer cells in mediating anti-plasmodial activity of the CD8+ T cells. In order to generate a PyCS-specific CD8+ T-cell line from these K<sup>d</sup> Tg mice, we had tried different prime–boost immunization regimens, i.e., a combination of immunizations with a recombinant adenovirus expressing PyCS, a whole irradiated PySpz in addition to SYVPSAEQI peptide, unsuccessfully. It was also documented in our previous research that CD11c-K<sup>d</sup> Tg or CD68-K<sup>d</sup> Tg mice elicited much fewer number of PyCSspecific CD8 T cells *in vivo* than that of MHC-I-K<sup>d</sup> mice after immunization with either SYVPSAEQI peptide or recombinant adenovirus expressing PyCS (Huang et al., 2013). The maximum number of PyCS-specific CD8+ T cells we could obtain from a group of three immunized K<sup>d</sup> Tg mice was 1 <sup>×</sup> 107 total cells after stimulating them 3–4 times *in vitro* with radiationattenuated, APCs derived from MHC-I K<sup>d</sup> Tg mice. The difficulty of expanding a PyCS-specific CD8+ T-cell line *in vitro* may be due to the lack of K<sup>d</sup> expression by CD8<sup>+</sup> T cells themselves for presenting the peptide to each other. In any case, we are currently attempting to breed and increase to a large number of CD11c-K<sup>d</sup> Tg or hCD68-K<sup>d</sup> Tg mice, so that we will be able to expand PyCS-specific CD8+ T cells *in vivo* before direct cell sorting in the future.

#### **ACKNOWLEDGMENT**

We thank Dr. Ruth Nussenzweig and Dr. Victor Nussenzweig for their supports and Dr. Vincent Sahi for assisting FACS analyses. We also thank the NIH Tetramer Core Facility at Emory University for generating and providing an Allophycocyanin-labeled H-2Kd/SYVPSAEQI-tetramer. This work was supported by a grant from NIH AI10289.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 September 2014; accepted: 20 January 2015; published online: 12 February 2015.*

*Citation: Huang J, Tsao T, Zhang M, Rai U, Tsuji M and Li X (2015) A sufficient role of MHC class I molecules on hepatocytes in anti-plasmodial activity of CD8*+ *T cells in vivo. Front. Microbiol. 6:69. doi: 10.3389/fmicb.2015.00069*

*This article was submitted to Microbial Immunology, a section of the jurnal Frontiers in Microbiology.*

*Copyright © 2015 Huang, Tsao, Zhang, Rai, Tsuji and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### No more monkeying around: primate malaria model systems are key to understanding Plasmodium vivax liver-stage biology, hypnozoites, and relapses

#### *Chester Joyner 1, John W. Barnwell 1,2 and Mary R. Galinski 1,3 \**

<sup>1</sup> Malaria Host–Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA

<sup>2</sup> Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA, USA

<sup>3</sup> Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, GA, USA

#### *Edited by:*

Ute Frevert, New York University School of Medicine, USA

#### *Reviewed by:*

Hajime Hisaeda, Gunma University, Japan

Dominique Mazier, University Pierre and Marie Curie, France

#### *\*Correspondence:*

Mary R. Galinski, Malaria Host-Pathogen Interaction Center, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329, USA e-mail<EMAIL_ADDRESS>
Plasmodium vivax is a human malaria parasite responsible for significant morbidity worldwide and potentially death. This parasite possesses formidable liver-stage biology that involves the formation of dormant parasites known as hypnozoites. Hypnozoites are capable of activating weeks, months, or years after a primary blood-stage infection causing relapsing bouts of illness. Elimination of this dormant parasitic reservoir will be critical for global malaria eradication. Although hypnozoites were first discovered in 1982, few advancements have been made to understand their composition and biology. Until recently, in vitro models did not exist to study these forms and studying them from human ex vivo samples was virtually impossible. Today, non-human primate (NHP) models and modern systems biology approaches are poised as tools to enable the in-depth study of P. vivax liver-stage biology, including hypnozoites and relapses. NHP liver-stage model systems for P. vivax and the related simian malaria species P. cynomolgi are discussed along with perspectives regarding metabolite biomarker discovery, putative roles of extracellular vesicles, and relapse immunobiology.

#### **Keywords: malaria,** *Plasmodium***,** *P. vivax***,** *P. cynomolgi***, liver, hypnozoite, dormancy, non-human primate animal models**

Malaria is responsible for significant morbidity, mortality, and socioeconomic hardships in about 100 countries (World Health Organization, 2014). The causative agents of the disease are parasitic protists of the genus *Plasmodium*, which have a complex life-cycle involving a vertebrate and invertebrate host. After infecting susceptible mammals, the parasite undergoes obligate, clinically silent development in the liver prior to entering the blood and causing the clinical symptoms and pathology associated with malaria. Neutralizing the parasites in the liver has been a goal to prevent blood-stage infection and, therefore, clinical disease and transmission. Indeed, targeting liver stage forms (LSFs) has been a strong theme in current anti-malarial drug and vaccine efforts (Abdulla et al., 2011; Duffy et al., 2012). Moreover, preventing relapse infections is especially important in light of research demonstrating that the majority of *Plasmodium vivax* malaria episodes are due to relapses, which result from the activation of dormant forms in the liver, and not from new, mosquito-borne infections (Betuela et al., 2012; White et al., 2014).

Naturally, human clinical studies relating to the biology of LSFs and host–pathogen interactions in the liver are prohibitive, and regardless, influenced by uncontrollable variables; e.g., diet and medications. Unlike human studies, experimental NHP model systems are well suited for studying LSFs and relapse biology. Future malaria research with non-human primates (NHPs) on these topics will undoubtedly include large-scale'omics,' advanced immune profiling, mathematical modeling, computational biology, and the integration of clinical and 'omics' datasets (Galinski et al., 2013, 2014; Voit, 2013).

While research using NHPs will inevitably remain limited worldwide within a few capable biomedical research centers, such investigations with the public release of datasets will enable many more investigators to participate in associated areas of research and development. Moreover, collaborations with investigators at these centers can lead to new research directions including much needed translational studies to improve diagnostics and clinical care and to develop and test new anti-malarial interventions and vaccine candidates. From our perspective, these factors make NHP model systems critical to advancing the world toward malaria eradication.

#### **NHP-MALARIA MODELS OVERVIEW**

Non-human primate model systems have been instrumental in malaria research for decades whether for furthering basic understanding of *Plasmodium* biology, malaria pathogenesis, or preclinical investigations pertinent to developing new interventions (Coatney et al., 1971; Collins, 1974; Galinski and Barnwell, 2012; Beignon et al., 2014). Notably, they were critical for the discovery of dormant forms of the parasite in the liver, known as hypnozoites. These forms were first discovered in rhesus macaques (*Macaca mulatta*) experimentally infected with *P. cynomolgi* (Krotoski et al., 1982b), and then in chimpanzees infected with *P. vivax* (Krotoski et al., 1982a). Research from the field has confirmed that hypnozoites can stay dormant for weeks, months, or years after a primary infection and then activate and result in relapses, with new cycles of blood-stage parasitemias and illness (White and Imwong, 2012). Recently, NHPs were critical for the development of an *in vitro*, primary hepatocyte culture system that

supported the cultivation of *P. cynomolgi* LSFs for approximately 40 days and provided the first tangible evidence that hypnozoites existed and were capable of activating and multiplying to generate merozoites (Barnwell and Galinski, 2014; Dembele et al., 2014).

Various NHP-simian and human malaria parasite combinations can be used to study *Plasmodium* biology. Many strains of the parasites that infect NHPs, including four validated relapsing species, are available and can be used to address scientific questions relating to LSF biology (**Table 1**). Different strains of *P. cynomolgi* and *P. vivax* that possess distinctive relapse patterns can be utilized to study the consequences of frequent versus infrequent relapses on the host immune system. A suitable mouse model is not currently available to study such phenomena. Indeed, humanized mice containing human hepatocytes have been demonstrated to support *P. falciparum* liver-stage growth (Vaughan et al., 2012; Kaushansky et al., 2014). These models also appear to have some utility for *P. vivax* because they appear to support the development of hypnozoites (Mikolajczak et al., 2013). However, these mice lack intact immune systems and, thus, are deficient when addressing immunobiological questions, whether for *P. falciparum* or other primate malaria species (Kaushansky et al., 2014).

#### **VIVAX MALARIA – NHP MODELS**

While NHP models have been used occasionally to supplement fundamental *P. falciparum* research findings from culture systems and for pre-clinical studies, *P. vivax* research over the last few decades would have been virtually impossible without NHP models; i.e., *Aotus* and *Saimiri* monkeys (Galinski and Barnwell, 2012). Unlike *P. falciparum*, a long-term *in vitro* culture system for *P. vivax* does not currently exist due to the need for a regular supply of reticulocytes (Noulin et al., 2013), and thus, we believe yet to be defined culture media components that better mimic the host environment are also needed (unpublished data). In the meantime, NHP models have been critical for generating *P. vivax* material for in-depth analyses (Anderson et al., 2015) and NHP experimental studies continue to complement and expand upon blood-stage analyses that are now possible with small clinical samples attained from human infections (Russell et al., 2012; Galinski et al., 2014).

To investigate hypnozoites and relapses*, Aotus* or *Saimiri*species can be infected with NHP-adapted *P. vivax* strains via mosquito inoculation or syringe injection of sporozoites into a blood vessel (**Table 1**; Galinski and Barnwell, 2012; Galinski et al., 2013). Similar to human infections, relapsing, recrudescing, or chronic infection profiles can be observed in these models provided the animals are splenectomized to interfere with an overly robust removal of infected erythrocytes (**Figure 1**). In contrast to relapses, recrudescences are the result of untreated or persistent blood-stage infections that become sub-patent, below the level of detection by microscopy, followed by an eventual return to patency; such recurring parasitemias are distinct from relapse parasitemias that are due to the activation of hypnozoites and release of a new brood of merozoites from the liver.

Blood-stage parasitemias, which begin to develop within 8–10 days, can be curatively treated without destroying the hypnozoites. PCR testing can confirm the absence of blood-stage parasites, and thus, any subsequent blood-stage infections can be confirmed as relapses and not recrudescences. This experimental strategy is currently the only reliable means to study vivax relapses, with the caveats that these animals are small (typically about 1 kg), parasitemia is typically low or moderate (1–2%), and only small blood volumes can be taken (6 ml/kg/month) based on Institutional Animal Care and Use Committee (IACUC) guidelines. One strain in particular (named the Brazil VII strain) is being developed at the Centers for Disease Control and Prevention (CDC) for studying relapses as it shows multiple relapse patterns over a period of several months similar to that observed previously in humans with "tropical strains" (**Table 1**).

#### **SIMIAN PARASITE – NHP MODELS FOR VIVAX MALARIA**

Simian malaria parasite-NHP models are powerful systems to investigate LSF biology, hypnozoites, and relapses compared to the small New World NHPs. Simian malaria parasites productively infect Old World monkeys, including rhesus macaques (*M. mulatta*) and long-tailed macaques (*M. fascicularis*), which possess similar genetic composition and physiology to humans (Gardner and Luciw, 2008; Messaoudi et al., 2011; Zimin et al., 2014). The macaques are much larger than New World monkeys, which allows for greater blood or bone marrow draws (up to a maximum of 10 ml/kg/month) for isolation of parasite material and host cells for immunobiological studies. Additionally, more reagents exist for experimentation with these NHP species. Furthermore, large amounts of liver-material can be collected via biopsies or whole livers to isolate LSFs for downstream experiments.

*Plasmodium cynomolgi* is a "sister species" of *P. vivax.* These closely related parasites share similar biology such as the formation of hypnozoites and caveolae vesicle complexes in infected erythrocytes (Aikawa et al., 1975; Akinyi et al., 2012; Tachibana et al., 2012). Tens of millions of *P. cynomolgi* sporozoites can be generated in a specified, experimental time-frame compared to *P. vivax* sporozoites which are more difficult to generate (Rosenberg and Rungsiwongse, 1991; unpublished data). They can then be used to experimentally infect macaques and lead to productive blood-stage parasitemias (Collins et al., 1999). Furthermore, the same experimental approach used in Small New World Monkey infections with *P. vivax* can be employed to study relapses (see Vivax Malaria – NHP Models). Two other simian malaria species that could be useful for studying hypnozoites or relapse mechanisms are *P. fieldi* and, especially, *P. simiovale* (**Table 1**).

As with *P. vivax*, many *P. cynomolgi* isolates exist that have their own infection and relapse characteristics (**Table 1**). The number and frequency of relapses in rhesus infected with different strains of *P. cynomolgi* can be predicted with greater accuracy than typically possible with New World monkey-adapted *P. vivax* isolates although *P. vivax* Brazil VII has so far been uniquely dependable in this regard (unpublished data). In rhesus, variables such as the sporozoite inoculum can be altered to produce consistent infection patterns even though inter-individual variability between animals as well as inherent properties of the strain being used in the study can influence infection kinetics (Schmidt, 1986).

*Plasmodium cynomolgi* can be genetically manipulated more easily than *P. vivax,* though *P. vivax* transfection has been accomplished at the CDC and Emory, where transient transfection

**Table 1 |**

*Plasmodium*

**species and strains with critical**

**characteristics**

 **for studying**

**liver-stages.**

of trophozoites was demonstrated in *Saimiri boliviensis* (Pfahler et al., 2006). In recent years, we have developed *P. cynomolgi* parasites with integrated transgenes, including a *red fluorescent protein* (*rfp*) gene (Akinyi et al., 2012; unpublished data). Transient RFP- and green fluorescent protein-expressing *P. cynomolgi* parasites have also been reported and used to purify *P. cynomolgi* LSFs from NHP primary hepatocyte cultures using fluorescenceactivated cell sorting (Voorberg-van derWel et al., 2013). Technical hurdles such as achieving better yields of purified parasites for downstream experiments still remain. Nonetheless, these are monumental breakthroughs given the challenges working with these parasites.

#### **PRIMARY NHP HEPATOCYTE CULTURES**

NHP primary hepatocyte cell cultures are valuable for exploratory studies and may prove to be crucial for validating *in vivo* experiments as well as mechanistic studies requiring gene knockdowns, drug treatment, etc. Critical advancements have been made toward optimizing *in vitro* studies with *Plasmodium* LSFs, particularly with *P. cynomolgi* and rhesus monkey primary hepatocytes, even though infection rates, and thus, parasite yields still remain low (Voorberg-van der Wel et al., 2013). The most critical advancement has been the establishment of a long-term culture system using primary NHP hepatocytes that can support *P. cynomolgi* hypnozoites and other LSFs for up to 40 days; importantly, this system allows for hypnozoites to activate and develop into schizonts capable of releasing merozoites (Barnwell and Galinski, 2014; Dembele et al., 2014). This breakthrough ramps up research in this area and provides a workable system for validating *in vivo* findings.

#### **SYSTEMS BIOLOGY APPROACHES**

Systems biology approaches have been recently pioneered to study infectious diseases and vaccine efficacy (Li et al., 2014; Petrizzo et al., 2014; Zak et al., 2014). Systems biology foregoes traditional reductionist approaches and focuses on a biological system in its entirety. Typically, multiple 'omics technologies are employed to generate large datasets and methods are developed to integrate those datasets. Advanced mathematics and statistics are required to generate computational models of specific biological processes, such as hematopoiesis, immune responses, and infectious disease pathogenesis. Indeed, these strategies are being utilized by the Malaria Host–Pathogen Interaction Center (MaHPIC) using Old World and New World monkey models to study malaria and bring a wealth of data and novel results to the research community via online resources. We believe that systems biology approaches will likewise have utility for studying *Plasmodium* LSFs and the mechanisms behind relapses.

Systems biology methods can be attempted to study LSFs using transgenic, fluorescent LSFs isolated by fluorescence-activated cell sorting from *ex vivo* liver tissue. We and others (Voorberg-van der Wel et al., 2013) hope to demonstrate that adequate, purified *ex vivo* parasite material for downstream experimentation can be attained using such strategies. Performing transcriptomic, proteomic, and metabolomic analyses on the *ex vivo* material holds potential for identifying biochemical and molecular pathways important in hypnozoite biology. A hypnozoite proteome may help elucidate new vaccine candidates whereas transcriptome and metabolome data could give insight into other potential biochemical and molecular pathways that could become drug targets. Indeed, headway is being made in understanding hypnozoite biology with the recent demonstration that epigenetic programming could be responsible for latency (Dembele et al., 2014). Notably, however, modifying epigenetic changes *in vivo* may have detrimental side effects to the host organism, and thus, this may not be a feasible treatment strategy in humans.

We are well aware of the scientific and physiological challenges, but also optimistic that highly sensitive systems biology approaches that include metabolomics can help identify biomarkers that could predict the presence of hypnozoites and serve as diagnostic tools. Metabolomics is a relatively new, yet powerful, scientific discipline that is gaining traction in the fight against malaria (Olszewski et al., 2009; Kafsack and Llinás, 2010; Lakshmanan et al., 2011; Salinas et al., 2014). It is exciting to consider how high-resolution mass spectrometry could potentially identify a metabolic biomarker(s) (predictably of host origin) in the serum, plasma, urine, or saliva that is indicative of the presence of LSFs and the need for treatment. Potential biomarkers are best sought from an *in vivo* infection where host–parasite interactions can be investigated in the context of the normal physiology of the parasite and the host. Experimental NHP models can be informative in this regard. Diets and other variables can be controlled, and infected samples can be collected daily, or even multiple times daily if useful, over the course of a designated infection period through multiple relapse episodes without the immediate, ethical need for treatment.