Critical Roles for LIGHT and Its Receptors in Generating T Cell-Mediated Immunity during Leishmania donovani Infection

LIGHT (TNFSF14) is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTβR). We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL) caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTβR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4+ T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.


Introduction
Tumour necrosis factor (TNF) superfamily members are involved in many biological functions, including cell growth and differentiation, apoptosis and organogenesis [1]. This broad range of activities is achieved by TNF family members interacting with functional receptors associated with distinct cell signalling pathways [2]. TNF, lymphotoxin (LT)a, LTb and LIGHT (TNFSF14) comprise a closely related set of ligands in the TNF family [3,4]. TNF exists as a cell-bound or soluble homotrimer that binds TNF receptor (TNFR)1 and TNFR2 [5,6]. LTa can form a soluble homotrimer (LTa 3 ) that binds TNFR1, TNFR2 and HVEM [5,7], but can also form a cell-bound heterotrimer with LTb (LTa 1 b 2 ) that binds and signals through LTbR [8]. LIGHT exists in cell-bound and soluble forms that interact with both LTbR and herpes virus entry mediator (HVEM) [7,9,10]. HVEM also engages members of the immunoglobulin superfamily; B and T lymphocyte attenuator (BTLA) [11] and CD160 [12], as well as the envelope glycoprotein D of Herpes Simplex virus [13]. HVEM activates BTLA inhibitory signalling via SHP phosphatases suppressing T cell activation [14]. LIGHT, LTa and the Ig superfamily ligands can also activate HVEM-dependent cell survival signalling via NF-kB [15].
LIGHT has emerged as a key mediator of inflammation and immune homeostasis [4,14]. There is broad expression of LIGHT and HVEM in the hematopoietic compartment [7,9,16,17,18], while LTbR expression is largely restricted to stromal and myeloid cells [7,19,20]. LTbR and HVEM are implicated as key host defence mechanisms against persistent viral [21] and bacterial pathogens [22]. However, little is known about the role of these receptors in infection with parasites that establish persistent infections in their hosts.
The protozoan parasite Leishmania donovani causes persistent infections in humans and experimental animals [23,24]. We and others have defined important roles for TNF and LTa in host resistance in a mouse model of visceral leishmaniasis (VL) caused by L. donovani [25,26,27]. This disease model is characterised by an acute, resolving infection in the liver involving the formation of proinflammatory granulomas around infected Kupffer cells, and the establishment of a chronic infection in the spleen (reviewed in [24,28,29,30]). Mice deficient in TNF are highly susceptible to L. donovani infection, and die in the second month of infection with unchecked parasite growth [25,26,31]. However, TNF also induces disease pathology in the spleen, including the loss of marginal zone macrophages and down-regulation of chemokine receptor expression by dendritic cells (DCs) [31,32]. Mice lacking LTa display a less severe phenotype characterised by disrupted cellular trafficking into the liver and reduced control of hepatic parasite growth, although ultimately, infection is resolved in this organ [26].
Here we investigated the impact of L. donovani infection in LIGHT-deficient mice, as well as the roles of LIGHT binding each of its functional, cognate receptors during infection. We report a critical role for LIGHT in the resolution of hepatic infection, and more specifically, identify an important role for LIGHT-HVEM interactions in stimulating IL-12 production by DCs, and hence in the control of parasitic infections. Conversely, we also discovered that blockade of LIGHT-LTbR interactions dramatically enhanced early anti-parasitic immunity. Thus, we have identified distinct and opposing roles for LIGHT engagement of each of its receptors during infection.

Organ-specific expression of LIGHT in response to L. donovani infection
Homeostatic levels of LIGHT mRNA in liver ( Figure 1A) and spleen ( Figure 1B) differed by an order of magnitude in naïve mice. Following L. donovani infection, LIGHT mRNA accumulation increased in the liver over the first 28 days, and remained elevated despite infection largely resolving ( Figure 1C). In contrast, the initially high splenic LIGHT mRNA levels decreased over the first 28 days of infection ( Figure 1B), and remained diminished as a persistent L. donovani infection became established ( Figure S1A). Thus, an organ-specific pattern of LIGHT mRNA expression emerged in response to L. donovani infection.
To establish whether LIGHT was required to control infection, we infected LIGHT-deficient and control C57BL/6 mice with L. donovani and followed the course of infection in the spleen and liver for 90 days. Despite no difference in hepatic parasite burdens in the first 7 days of infection, parasite growth was significantly greater in the livers of LIGHT-deficient mice from day 14 p.i. onwards. Furthermore, these mice failed to fully resolve hepatic infection in the time period studied ( Figure 1C). TNF, IFNc and nitric oxide (measured as the surrogate marker inducible nitric oxide synthase; NOS2) are all critical for control of L. donovani in the liver [26,27,31,33,34]. Serum TNF and IFNc levels were reduced, and the accumulation of hepatic NOS2, IFNc and TNF mRNA were all lower in LIGHT-deficient mice at 14 days, compared with control animals ( Figure S1B-E). In the spleen, there were no significant differences in parasite burdens between C57BL/6 and B6.LIGHT 2/2 mice at any time point studied (data not shown). The accumulation of NOS2 mRNA was much lower in the spleen of C57BL/6 and B6.LIGHT 2/2 mice at day 14 p.i., compared with the liver, and no difference in IFNc, TNF and NOS2 mRNA accumulation in the spleen between mouse strains was observed at this time point (Figure S1B-E). We therefore focused our attention on the liver.
The formation of pro-inflammatory granulomas around infected Kupffer cells is a critical step in host control of parasite growth in the liver [24,28,29,30]. Liver immunohistochemistry revealed an increased number of inflammatory foci associated with increased parasite burden and impaired formation of inflammatory granulomas in B6.LIGHT 2/2 mice at day 14 p.i., relative to control mice, as indicated by a greater frequency of infected Kupffer cells with no surrounding leukocytes (KC), and a lower frequency of immature (IG) and mature granulomas (MG) (Figure 2A). To ensure that the failure to develop anti-parasitic immunity in the liver did not result from an as yet unidentified developmental defect in LIGHT-deficient mice, BM chimeras were made by engrafting LIGHT-deficient or control (C57BL/6) BM cells into lethally irradiated C57BL/6 mice. These mice were infected and parasite burdens measured 14 days later. BM chimeric mice responded to hepatic infection in accordance with their source of BM ( Figure 2B). Hepatic parasite burdens were significantly increased in LIGHT-deficient BM chimeras compared to controls, indicating that LIGHT production by leukocytes was required for the efficient generation of anti-parasitic immunity in the liver at this early time point in infection. Additional experiments in T and B cell-deficient B6.RAG1 2/2 mice receiving LIGHT-deficient or wild-type T cells showed that LIGHT production by T cells was not required for the development of anti-parasitic immunity in the liver during the first 14 days of infection ( Figure 2C). However, we cannot exclude a role for T cell-derived LIGHT in the generation of optimal early host immunity following L. donovani infection because we did find a small, but significant difference (p,0.001) in parasite burden at day 14 p.i. between B6.RAG1 2/2 mice that received LIGHT-deficient T cells and those that received wild-type T cells ( Figure 2C). We also found that T cells per se were not a major source of hepatic LIGHT mRNA, although their presence was required for LIGHT expression to increase in the liver during this early period of infection ( Figure 2D). Together, these data indicate that the generation of immune responses against L. donovani in the liver were impaired in the first 14 days of infection the absence of LIGHT.
Treatment with anti-HVEM mAb impairs parasite clearance while treatment with anti-LTbR mAb promotes this process Treatment with anti-HVEM mAb (LH1) that blocks LIGHT binding to HVEM, but not HVEM-BTLA interactions [35], significantly increased hepatic parasite load at day 14 p.i. in mice, similar to the increase in parasite burden observed in LIGHTdeficient mice ( Figure 3A). Surprisingly, hepatic parasite burdens were significantly decreased by treatment of mice with anti-LTbR

Author Summary
Visceral leishmaniasis (VL) is a potentially fatal human disease caused by the intracellular protozoan parasites Leishmania donovani and L. infantum (chagasi). Parasites infect macrophages throughout the viscera, though the spleen and liver are the major sites of disease. VL is responsible for significant morbidity and mortality in the developing world, particularly in India, Sudan, Nepal, Bangladesh and Brazil. Because of the intrusive techniques required to analyse tissue in VL patients, our current understanding of the host immune response during VL largely derives from studies performed in genetically susceptible mice. We have discovered that mice which are unable to produce a cytokine called LIGHT have poor control of L. donovani infection in the liver, compared with wild-type control animals. In addition, we demonstrated that LIGHT has distinct roles during VL, depending on which of its two major cell-bound receptors it engages. Finally, we identified an antibody that stimulates the lymphotoxin b receptor (one of the LIGHT receptors), that can stimulate anti-parasitic activity during an established infection, thereby identifying this receptor as a therapeutic target during disease. mAb (LLBT2) that blocks LIGHT binding to LTbR, but not LTa 1 b 2 -LTbR interactions [35] ( Figure 3A). Antibody treatments had no significant effect on the low splenic parasite burden at this time point ( Figure S2). The formation of granulomas was significantly impaired by anti-HVEM mAb, as indicated by a greater frequency of KC and a lower frequency of IG and MG (p,0.05, k 2 analysis; Figure 3B). In contrast, granuloma formation was significantly enhanced by anti-LTbR mAb, as indicated by a lower frequency of KC and a higher frequency of IG and MG (p,0.05, k 2 analysis; Figure 3B). Thus, these results indicate that HVEM and LTbR have distinct and opposing roles during the first 14 days of infection.

Stimulation of LTbR improves parasite clearance during an established infection
To further investigate the role of LTbR in VL, we treated mice with the agonist anti-LTbR antibody (3C8) which blocks binding of both LTa 1 b 2 and LIGHT to LTbR, yet functions as an agonist directly activating LTbR signalling pathways [36,37]. The anti-LTbR 3C8 enhanced parasite clearance in the liver during an established infection ( Figure 4A), but had no anti-parasitic effect in the first 14 days of infection (data not shown), unlike the anti-LTbR mAb LLBT2 ( Figure 3A). Importantly, 3C8 also prevented parasite growth in the spleen between days 14 to 28 p.i. ( Figure 4B). In contrast, treatment with LLTB2 during established infection (days 14-28 p.i.) had no effect on parasite clearance in the liver or spleen (data not shown). Thus, treatment of L. donovaniinfected mice with two different anti-LTbR mAbs had distinct effects on the course of infection, reflecting different functional properties of these mAbs.

Treatment with anti-HVEM mAb impairs Th1-mediated immunity
We next sought to identify anti-parasitic mechanisms dependent upon LIGHT-HVEM signalling. We previously showed that early Figure 1. LIGHT is required for efficient parasite clearance in the liver. C57BL/6 mice were infected with L. donovani, and LIGHT mRNA accumulation was measured in liver (A) and spleen (B) at indicated times (expressed as the number of LIGHT mRNA molecules per 1000 HPRT mRNA molecules). (C) C57BL/6 (closed squares) and B6.LIGHT 2/2 (open squares) mice were infected with L. donovani, and hepatic parasite burdens were measured from day 7 p.i. to day 90 p.i. and data represent the mean +/2SEM. One representative experiment of at least 2 performed is shown (n = 4-5 mice per treatment group in each experiment). Statistical differences of p,0.05 (*), p,0.01 (**) or p,0.001 (***) are indicated. doi:10.1371/journal.ppat.1002279.g001 splenic IL-12/IL-23p40 production by DC is critical for the efficient generation of immunity in the liver [38,39]. Although no change in IL-12p35 mRNA accumulation was observed in any treatment group, the anti-HVEM mAb (LH1) inhibited splenic DC IL-12/IL-23p40 mRNA accumulation in response to L. donovani infection ( Figure 5A). We next evaluated the importance of LIGHT-HVEM co-stimulatory signals for the development of L. donovani-specific CD4 + T cell priming and Th1 differentiation, the latter being a known IL-12-dependent process [39,40,41]. Mice were injected with CFSE-labelled OVA-specific CD4 + (OT-II) T cells, then infected with transgenic OVA-expressing L. donovani [42], and antigen-specific CD4 + T cell proliferation was assessed 4 days later by CFSE dilution. No antigen-specific CD4 + T cell proliferation was observed when mice were infected with wild-type parasites ( Figure 5B), so no bystander activation had occurred, and OT-II cell proliferation occurred equally in control and anti-HVEMtreated mice ( Figure 5B), indicating that LIGHT-HVEM interactions were not required for early priming of CD4 + T cell proliferation. In support of this result, proliferation of polyclonal antigen-specific, CD4 + T cells was similar between cells isolated from the spleens of control-treated and anti-HVEM treated mice ( Figure 5C). However, production of IFNc and TNF by these antigen-specific CD4 + T cells was inhibited by anti-HVEM mAb ( Figure 5C). Furthermore, direct ex vivo production of IFNc by hepatic CD4 + T cells (both total number and frequency) was significantly reduced by anti-HVEM mAb ( Figure 5D), indicating that LIGHT-HVEM interactions play an important role in generating Th1 cell responses following L. donovani infection.

Treatment with anti-LTbR mAb promotes Th1-mediated immunity
The anti-LTbR mAb (LLTB2) inhibited parasite growth in acute experimental VL ( Figure 3A), but had no effect on splenic DC IL-12/IL-23p40 or IL-12p35 mRNA accumulation at 5 hours p.i. (Figure 6A), and no effect on the expansion of OVA-specific CD4 + T cells (OT-II cells) in mice infected with OVA-transgenic L. donovani ( Figure 6B). We also found no differences in antigenspecific recall responses in splenic CD4 + T cells isolated from infected mice on day 14 p.i., yet the amount of TNF and IFNc produced upon antigen-specific CD4 + T cell stimulation was greatly enhanced in these cells from mice treated with anti-LTbR mAb ( Figure 6C). In addition, the number and frequency of IFNcproducing hepatic CD4 + T cells measured directly ex vivo on day 14 p.i. was significantly increased in these mice ( Figure 6D), suggesting LIGHT-LTbR binding suppresses the development of Th1 cell responses in VL.

Treatment with anti-LTbR mAb promotes parasite clearance in the liver early during infection
We next investigated timing requirements for treatment with the anti-LTbR mAb (LLTB2) during acute infection with L. donovani. A single dose (100 mg) of anti-LTbR mAb at the time of infection was sufficient to reduce hepatic parasite burden as early as day 7 p.i. (Figure 7A). To test whether treatment with anti-LTbR mAb was simply shunting available LIGHT onto HVEM, we also co-treated mice with anti-LTbR (LLTB2) and anti-HVEM (LH1) mAbs ( Figure 7B), and found no additional effect of coadministration over anti-LTbR alone by day 7 p.i., indicating that increased, early anti-parasitic immunity observed after anti-LTbR mAb (LLTB2) treatment was not caused by enhanced HVEMmediated co-stimulation. Of note, there was no effect of anti-HVEM mAb treatment alone at day 7 p.i., indicating that the effect of this treatment on parasite burden only becomes apparent between days 7-14 p.i., similar to what was observed in LIGHTdeficient mice ( Figure 1C). To test whether anti-LTbR mAb (LLTB2) agonist activity might account for the above effect, we treated LIGHT-deficient mice with this antibody and measured liver parasite burdens at day 7 p.i. (Figure 7C). Although a significant reduction in parasite burden was found in C57BL/6 mice treated with anti-LTbR mAb (LLTB2), no such effect was observed in LIGHT-deficient mice, indicating that the likely mechanism of action was via the blockade of LIGHT binding LTbR.

Enhanced parasite clearance observed following treatment with anti-LTbR mAb requires CD4 + T cells and TNF
We investigated the cellular requirements for the early antiparasitic effects of anti-LTbR mAb (LLTB2). Treatment with anti-LTbR mAb had no impact on hepatic parasite burdens in B6.RAG1 2/2 mice at day 7 p.i. (Figure S3), suggesting that B and/or T lymphocytes are required for the enhanced parasite clearance resulting from this treatment. We focused our attention on T cells because we have previously shown that B cells play a negative regulatory role in the liver during infection [43]. Depletion of CD4 + or CD8 + T cells alone during the first 7 days of infection had no effect on hepatic parasite burden ( Figure 8A), despite T cells being required for the control of parasite growth at later stages of infection [26,44,45]. However, depletion of CD4 + T cells, but not CD8 + T cells, prevented the anti-parasitic effect mediated by anti-LTbR at day 7 p.i. (Figure 8A). Given that NKT cells comprise a significant proportion of hepatic CD4 + T cells, we also investigated whether this cell subset was required for the increased anti-parasitic activity. Treatment of NKT cell-deficient (B6.Ja18 2/2 ) mice with the anti-LTbR mAb ( Figure 8A) had no impact on the decreased liver parasite burden, indicating that conventional CD4 + T cells, but not NKT cells, are required for the enhanced parasite clearance following anti-LTbR mAb treatment.
We observed increased CD4 + T cell TNF and IFNc production was associated with improved control of parasite growth resulting from anti-LTbR mAb treatment ( Figure 6). We next assessed whether these cytokines were required for the enhanced parasite clearance in mice receiving anti-LTbR (LLTB2) mAb. Hepatic parasite burdens were decreased similarly in anti-LTbR mAb treated control and IFNc-deficient mice ( Figure 8B). However, anti-LTbR mAb treatment in TNF-deficient mice had no impact on hepatic parasite burden ( Figure 8B), indicating that TNF is critical for this enhanced parasite clearance. The failure of anti-LTbR mAb treatment in TNF-deficient animals was not caused by reduced expression of LTbR on the cells of these mice, as LTbR expression levels were no different to those on immune cells from C57BL/6 control mice (data not shown). Furthermore, adoptive transfer of wild type and TNF-deficient CD4 + T cells into B6.RAG1 2/2 mice and treatment with anti-LTbR mAb demonstrated that CD4 + T cells did not have to produce TNF ( Figure 8C). Thus, anti-LTbR mAb treatment increased early hepatic anti-parasitic immunity by mechanisms requiring conventional CD4 + T cells and TNF, the latter potentially coming from a non-T cell source.

Discussion
We have identified distinct and opposing roles for LIGHT and its receptors during infection. LIGHT has important roles in T cell costimulation [3,14]. Blockade of LIGHT impairs allogeneic T cell responses and graft versus host disease [46,47], while overexpression of LIGHT by T cells causes inflammatory disease of the gut and reproductive tissues [48,49]. Our results indicate that these effects could be mediated via the LIGHT-HVEM axis between T cells and DC. Early DC IL-12 production depends on the presence of T cells, and this IL-12 production is critical for generating anti-parasitic immune mechanisms that control L. donovani growth [24,39,40,50]. Our finding that anti-HVEM mAb blocks IL-12/IL-23p40 mRNA accumulation during infection is consistent with a previous study that reported BM-derived DCs from LIGHT-deficient animals were impaired in their ability to produce IL-12 following activation in vitro [51]. This study also showed that blockade of LIGHT with soluble receptors in mice infected with L. major, a cause of cutaneous leishmaniasis, resulted in reduced IL-12 generation, associated with diminished CD4 + T cell IFNc production and increased parasite growth and disease.
Our finding that T cells did not have to produce LIGHT in order to promote anti-parasitic immunity, together with data from L. major infection in mice [51], support a model whereby DC-derived LIGHT interacts with T cell HVEM to promote DC IL-12 production.
The defect in anti-parasitic immunity observed in the absence of LIGHT was restricted to the liver, and not the spleen. The reason for this is unclear, but could relate to the requirement for cellular recruitment and granuloma development for control of parasite growth in the liver. Although increased tissue weight and cellular expansion are features of L. donovani infection in the spleen, organised inflammatory granulomas are rarely observed in this tissue [24,28,29,30]. Importantly, parasite growth is contained in the spleen after 1-2 months of infection rather than efficiently controlled, as occurs in the liver. Therefore, it is possible that different anti-parasitic immune mechanisms operate in these two tissue sites during experimental VL with different requirements for LIGHT.
The LIGHT-specific blocking mAbs we have employed (LH1 and LLTB2) have previously been shown to selectively block interactions between LIGHT and its receptors (HVEM and LTbR, respectively) [35]. However, we cannot exclude the possibility that they may trigger some receptor activation following engagement, and that this may contribute to biological effects we have observed. In addition, because these mAbs cause the selective blockade of LIGHT binding to their respective receptors, we cannot rule out that they promote alternative receptor-ligand interactions ( Figure 9A). For example, blocking LIGHT interacting with HVEM may allow HVEM to more readily engage BTLA on cells to increase inhibitory signals ( Figure 9B), as well as increased CD160 signalling. Similarly, blockade of LIGHT binding LTbR may allow greater amounts of LIGHT to bind HVEM, thereby reducing negative signalling between HVEM and BTLA and potentially promoting LIGHT-HVEM-mediated T cell co-stimulation ( Figure 9C). However, this latter possibility  seems unlikely in the current study given that co-administration of LH1 and LLTB2 resulted in improved control of parasite growth ( Figure 7B). Instead, LIGHT may send inhibitory signals via LTbR early during infection, although no such LTbR-mediated negative signalling pathway has yet been defined.
The anti-parasitic effects of anti-LTbR mAb (LLTB2) were observed when it was administered at the time of infection, but not in mice with an established L. donovani infection, suggesting that a mAb with similar functional characteristics would have limited therapeutic potential for treatment of VL. However, our finding that the defined agonist anti-LTbR mAb (3C8) improved the rate of parasite clearance in the liver and reduced parasite load in the spleen, not only demonstrated fundamentally different biological activities for LLTB2 and 3C8 mAbs, but also shows that LTbR activation can promote beneficial immune mechanisms during established infection. This agonist antibody has previously been shown to promote DC development and maturation in vivo [20,37], and this may explain the anti-parasitic effects observed after administration to L. donovani-infected mice because we have previously shown that DC adoptive transfer can improve control of parasite growth in infected mice [32]. Hence, the activation of anti-parasitic immune mechanisms by stimulation of LTbR represents a potential therapeutic strategy against chronic infectious diseases like VL. However, a better understanding of the functional characteristics of the different anti-LTbR mAbs will be required in order to better harness their therapeutic potential, including identification of the specific epitopes they recognise and signalling pathways they activate.
We previously reported increased monocyte recruitment into the spleen in an experimental model of cerebral malaria following treatment of mice with the anti-LTbR mAb (LLTB2), and that this treatment protected mice from disease [52]. Interestingly, no protection from experimental cerebral malaria was afforded by treatment with the anti-LTbR (3C8) mAb (Randall and Engwerda, unpublished), again emphasising the functional differences between LLTB2 and 3C8 anti-LTbR mAbs. An intriguing finding from our current studies was an increase in hepatic and splenic monocyte recruitment following anti-LTbR mAb LLTB2 treatment (CD11b + Ly6C hi cells; Figure S4A and B). Flow cytometry analysis revealed that monocytes, along with DCs (both cDC and pDC), and neutrophils expressed the highest levels of LTbR in the liver, as previously reported [19,20,37], and furthermore, that expression of LTbR did not appear to change significantly on any of these cells during the first 5 days of infection with L. donovani ( Figure S5). However, the increased monocyte recruitment was not necessary for improved early control of parasite growth in treated animals in the current study because mice lacking CCL2 that have an impairment in monocyte mobilisation [53], also had improved control of parasite growth following anti-LTbR (LLTB2) treatment at day 7 p.i. ( Figure  S4C). Although the early anti-parasitic effect of anti-LTbR (LLTB2) mAb appeared to involve blocking of LIGHT-LTbR interactions, as indicated by the failure of this antibody to improve parasite control in LIGHT-deficient mice ( Figure 7C), we cannot exclude the possibility that some effects of this antibody, such as increased monocyte mobilisation, might involve agonist activities. Regardless, given the important role for monocyte infiltration into sites of infection and tumour growth [54], our results indicate that manipulation of the LIGHT-LTbR signalling axis offers a potential way to improve monocyte mobilisation for therapeutic applications. Furthermore, given the recent report that monocytes can migrate into secondary lymphoid tissues in response to interactions with gram negative bacteria and/or their products, and then develop into CD209a + , CD206 + , CD14 + , CD11c hi DC capable of activating CD4 + T cells and cross-priming CD8 + T cells [55], our results suggest that manipulation of LIGHT signalling pathways may be one way to promote this process that may have applications in vaccination.
In summary, our findings further delineate the complex interactions between LIGHT and its receptors and demonstrate the therapeutic potential of modulating these immune regulatory pathways to improve disease outcomes. Our results provide mechanistic insight into the roles of LIGHT-HVEM interactions on DC function and CD4 + T cell priming, as well as anti-parasitic immune responses activated by blockade of LIGHT-LTbR interactions. Finally, we have identified two different mAbs that target LTbR with distinct functional outcomes on anti-parasitic immunity at different stages of infection.  [53] were bred at Monash University and maintained at the Queensland Institute of Medical Research. All mice used were age-and sex-matched (6-10 weeks), and were housed under specific-pathogen free conditions. Chimeric mice were prepared by irradiating B6.SJL.Ptprca mice with 11Gy and then engrafting with 3610 6 fresh bone marrow (BM) cells i.v. via the lateral tail vein. Mice were maintained on antibiotics for 2 weeks after engraftment and infected with L. donovani 8 weeks after receiving BM, as previously described [26]. Adoptive transfer of equal numbers (10 6 ) of purified CD4 + and CD8 + T cells (98% purity as determined by flow cytometry) into B6.RAG1 2/2 mice was performed as previously described [26].

Mice
MACS from naïve mice (hatched bars), untreated mice (grey bars), hamster IgG treated mice (open bars) or anti-HVEM treated mice (closed bars) at 5 hours p.i. (A). C57BL/6 mice treated with control hamster IgG or anti-HVEM mAb received CD45.1 + CFSE-labelled OVA-specific CD4 + T cells (OT-II) prior to infection with 2610 7 OVA-expressing L. donovani. OT-II CFSE dilution in the spleen was assessed by flow cytometry on day 4 p.i., after gating on CD45.1 + CD4 + TCRb + cells (B) and % CFSE dilution +/2 SEM is shown on each plot (CFSE dilution for 4 individual mice in each treatment group is shown, as well as CSFE dilution in a control animal that was infected with wild type parasites). Splenic CD4 + T cells were purified on day 14 p.i. from naïve mice or L. donovani-infected mice treated with control hamster IgG (open bars) or anti-HVEM mAb (closed bars) by positive MACS selection. CD4 + T cells were co-cultured with paraformaldehyde-fixed L. donovani amastigotes and irradiated spleen cells. Antigen-specific CD4 + T cell proliferation, as well as IFNc and TNF levels in cell culture supernatants were measured by cytometric bead array at 72 h post-stimulation (C). Hepatic CD4 + T cell (CD4 + TCRb + ) IFNc production was measured in L. donovani-infected mice treated with either control hamster IgG (solid line) or anti-HVEM (dashed line) mAb on day 14 p.i.. Representative histograms showing IFNc production by gated CD4 + TCRb + cells are shown and compared to isotype control antibody (solid gray) (D). Both the total number and frequency of IFNc-producing CD4 + T cells are shown graphically. One representative experiment of 3 performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p,0.05 (*), p,0.01 (**) or p,0.001 (***) for control versus mAb-treated mice are shown. doi:10.1371/journal.ppat.1002279.g005 Figure 6. Treatment with anti-LTbR mAb LLTB2 leads to increased antigen-specific CD4 + T cell IFNc and TNF production. DC IL-12/IL-23p40 mRNA accumulation (A), splenic OT-II CFSE dilution (B), antigen-specific splenic CD4 + T cell proliferation, IFNc and TNF production (C) and hepatic CD4 + T cell IFNc production (D) were measured as described in the legend of

Parasites and infections
L. donovani (LV9) and OVA-transgenic LV9 (PINK LV9) [42] were maintained by passage in B6.RAG1 2/2 mice and amastigotes were isolated from the spleens of chronically infected mice. Mice were infected by injecting 2610 7 amastigotes i.v. via the lateral tail vein, killed at the times indicated in the text by CO 2 asphyxiation and bled via cardiac puncture. In experiments examining DC IL-12/IL-23p40 production, mice were infected with 1610 8 amastigotes intravenously, as previously described [63]. Spleens and perfused livers were removed at times indicated and parasite burdens were determined from Diff-Quik-stained impression smears (Lab Aids, Narrabeen, Australia) and expressed as Leishman-Donovan units (LDU) (the number of amastigotes per 1,000 host nuclei multiplied by the organ weight in grams) [64]. Liver and spleen tissue were also preserved in either RNAlater (Sigma-Aldrich, Castle Hill, Australia) or Tissue-Tek O.C.T. compound (Sakura, Torrence, USA). Hepatic mononuclear cells and splenocytes were isolated as previously described [65].

Antibodies
All antibody-producing hybridomas were grown in 5% (v/v) foetal calf serum, RPMI containing 10 mM L-glutamine, 100 U/ ml penicillin and 100 mg/ml streptomycin. Purified antibody was prepared as previously described [63]. Mice were administered 100 mg of anti-LTbR mAb (LLTB2) or anti-HVEM mAb (LH1) [35] i.v. on the day of infection and every 5 days thereafter for 14 day experiments, or as a single dose on the day of infection for 7 day experiments.. The anti-LTbR mAb 3C8 was administered at 200 mg i.v. [36,37] starting at the times indicated in the text and every 5 days thereafter. The anti-LTbR mAb (LLTB2) or anti-HVEM mAb (LH1) specifically block the binding of LIGHT to either LTbR or HVEM, respectively, but do not disrupt interactions between these receptors and other functional ligands (i.e., LTa 1 b 2 for LTbR and BTLA for HVEM) [35]. The anti-LTbR mAb (3C8) blocks binding of both LTa 1 b 2 and LIGHT, but is an agonist directly activating LTbR [66]. Mice were depleted of CD4 + or CD8 + T cells with anti-CD4 (YTS191.1) or anti-CD8b (53-5.8) mAbs, respectively, as previously described [64]. Depletion of T cell subsets was confirmed at completion of experiments by assessing T cell numbers in the spleen by flow cytometry. Greater than 95% of CD4 + and CD8 + T cells were

CD4 + T cell proliferation
To assess antigen-specific T cell proliferation in vivo, mice were infected with OVA-transgenic PINK LV9 [42]. Splenic OVAspecific OT-II T cells were isolated and labelled with CFSE, as previously described [64]. CFSE-labelled OT-II cells (1610 6 ) were adoptively transferred into mice 2 h prior to infection with LV9 or PINK LV9. Expansion of CFSE + cells in the spleen was monitored by FACS 4 days later. In all of these experiments, control animals were included that received the same number of CFSE-labelled OT-II cells, but were infected with wild-type parasites. No OT-II proliferation was ever observed in these animals. Re-stimulation assays for endogenous splenic CD4 + T cells were performed as previously described [63].

Assessment of granuloma formation
The maturation of granulomas was scored around infected Kupffer cells in acetone-fixed liver sections as previously described [64,65].

Real Time PCR
RNA extraction and real-time RT-PCR was performed as previously described [63]. The number of IFNc, TNF, NOS-2, LIGHT and hypoxanthine phosphoribosyltransferase (HPRT) cDNA molecules in each liver tissue sample were calculated using Platinum Sybr Green Master Mix (Invitrogen Life Technologies) [63]. Standard curves were generated with known amounts of cDNA for each gene, and the number of cytokine molecules per 1000 HPRT molecules in each sample was calculated. The number of IL-12/IL-23p40 and IL-12p35 cDNA molecules in each DC sample were calculated using Taqman Gene Expression Assays (Applied Biosystems). Relative quantitation of gene expression was performed using the relative standard curve method as described by Applied Biosystems. Briefly, standard curves were prepared for all target and endogenous control genes using an uninfected control sample. HPRT was used as the endogenous control. The amount of target gene or endogenous control in each sample was calculated from the appropriate standard curves. The target amount was then divided by the endogenous control amount to give the normalized target value. The average normalized values for the four naïve samples were used as the calibrator.

Statistical analysis
Statistical differences between groups was determined using the Mann-Whitney U test using GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA) and p,0.05 was considered statistically significant. The distribution of hepatic histological responses was compared using X 2 analysis with Microsoft Excel software. All data are presented as the mean values plus or minus standard error unless otherwise stated.  Figure S5 Mononuclear cells isolated from the livers of naïve and L. donovani-infected mice at day 5 p.i. were phenotyped, labelled with a mAb against LTbR and enumerated by flow cytometry. Representative histograms gated on appropriate populations are shown for isotype control (solid grey shading), naïve mice (black line) and at day 5 p.i. with L. donovani (grey line). Cells were identified as follows: B cells (B220 + CD19 + ), cDC(CD11c hi MHCII hi ), monocytes (CD11b + Ly6c hi ), neutrophils (CD11b + Ly6c int ), NK cells (NK1.1 + TCRb 2 ), NKT cells (NK1.1 + TCRb + ), pDC (CD11c int 120G8 + ), CD4 + T cells (CD4 + TCRb + ), CD8 + T cells (CD8 + TCRb + ). Data presented is representative of 2 independent experiments. (TIF) Figure 9. Model of how manipulation of interactions between LIGHT and its receptors may impact on disease outcome. A. Under normal circumstances LIGHT will interact with HVEM and/or LTbR to promote anti-parasitic immunity, as well as with DCR3 (not shown). HVEM can also interact with BTLA to suppress immune responses, and can also interact with CD160 and HSV1 gD (not shown). LTa 1 b 2 can also bind to LTbR and provide signals essential for tissue organising and homeostasis. B. Blockade of LIGHT-HVEM with antibody will prevent HVEM-mediated costimulation and increase HVEM-BTLA-mediated inhibitory signals. LTbR-mediated inhibitory signals may also be increased via greater interactions with LIGHT. Together, this results in suppression of anti-parasitic immunity. C. Blockade of LIGHT-LTbR with antibody will potentially prevent LTbR-mediated inhibitory signals, but will also promote LIGHT-HVEM co-stimulatory signalling and reduce HVEM-BLTA inhibitory signals. Together, this stimulates increased anti-parasitic immunity. doi:10.1371/journal.ppat.1002279.g009