Deficiency of CD40 Reveals an Important Role for LIGHT in Anti-Leishmania Immunity

The outcome of infection with the intracellular pathogen Leishmania major, the causative agent of cutaneous leishmaniasis, is dependent on the nature of CD4⁺ Th cell subset that is induced (1–4). Resistance is usually correlated with the development of strong IFN-γ–producing CD4⁺ Th1 cells, which activate macrophages to produce NO, an effector molecule for killing intracellular parasites. In contrast, susceptibility has been associated with IL-4 and IL-10 production by Th2 cells, cytokines that deactivate macrophages and inhibit their intracellular parasite-killing ability (1, 5). A key factor that regulates the nature and quality of anti-Leishmania immunity in infected mice is the level of IL-12 production by dendritic cells (DCs) and the degree of responsiveness of naive CD4⁺ T cells to this cytokine (6–8). Thus, BALB/c mice are highly susceptible to L. major because their DCs produce very low amounts of IL-12, and their CD4⁺ T cells respond poorly to it because of the downregulation of IL-12Rβ expression (8–10). In contrast, DCs from the resistant C57BL/6 (B6) mice produce high levels of IL-12 and their CD4⁺ T cells maintain IL-12 responsiveness throughout infection (8). The pathways that influence early IL-12 production by DCs during L. major infection are still poorly understood, although the nature of activation and expression of various costimulatory molecules appear to be important (11, 12).

The TNF superfamily of cytokines and their receptors play critical roles in the development of the immune system and in immune regulation, inflammation, and autoimmunity by regulating cell death, survival, and differentiation (13–15). There are four closely related members of this family, which include lymphotoxin α (LTα, LTβ, TNF, and LIGHT. Their cognate receptors include TNFR1, TNFR2, lymphotoxin β receptor (LTβR), and herpes virus entry mediator (HVEM). Both LTα and LTβ are important for formation and structural integrity of peripheral lymphoid organs because mice with targeted deletion of these genes lack organized peripheral lymph nodes and their spleen architecture is structurally dysregulated (16–18). In contrast, LIGHT-deficient mice have normal peripheral lymphoid organs, suggesting that unlike LTα and LTβ, its function may be more closely related to immune modulation (16, 17). LIGHT is expressed on activated T cells, monocytes, granulocytes, and immature DCs (19–21) and binds to three receptors: LTβR, HVEM, and in humans, the decoy receptor, DcR3/TR6 (19, 22, 23). HVEM is expressed on resting T cells, NK cells, monocytes, immature DCs, and endothelial cells (22, 24, 25), whereas LTβR is expressed mostly on stromal cells, DCs, and endothelial cells but is absent on lymphocytes (26, 27). The interaction of LIGHT with its receptors modulates several biological processes including cell survival, inflammation, and upregulation of intracellular adhesion molecule-1 leading to tumor eradication (28). In addition, signals transmitted via LIGHT–HVEM interaction have costimulatory effects on T cells, enhancing their proliferation and cytokine production (19, 21). Thus, LIGHT expression enhances cytolytic T lymphocyte–mediated tumor immunity and allograft rejection (21, 29), and its overexpression on T cells results in extensive T cell–proliferative disorders that are characterized by massive polyclonal expansion of CD4⁺ and CD8⁺ T cells (30, 31).

We previously reported that blockade of LIGHT signaling results in severe impairment in anti-CD40, LPS-, and CpG-induced IL-12p40 production by macrophages and DCs in vitro and in vivo, and a concomitant impairment in CD4⁺ Th1 response and susceptibility to L. major in the otherwise resistant B6 mice (32). In this study, we extend these findings by showing that blockade of LIGHT interaction with its receptor leads to impaired DC maturation and IL-12 production post–L. major infection. We further demonstrate that LIGHT is required at both the priming and the maintenance stages of primary anti-Leishmania immunity but is completely dispensable during secondary immunity. Interestingly, blockade of LIGHT signaling in CD40-deficient (knockout [KO]) mice that healed their primary L. major infection after rIL-12 treatment led to lower delayed-type hypersensitivity (DTH) and higher parasite burden after secondary L. major challenge. Collectively, our results show that LIGHT is critical for priming and maintenance of CD4⁺ Th1 response during primary L. major infection but is completely dispensable for secondary immunity in intact wild type (WT) mice. However, in the absence of CD40 signaling, LIGHT becomes important for sustained IL-12 production, recall CD4⁺ Th1 response, and maintenance of secondary anti-Leishmania immunity.

Six- to 8-wk-old female B6 mice were purchased from Charles River Laboratories (St-Constant, QC). Female CD40-deficient (CD40 KO) and IL-12 KO mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen-free units at the Central Animal Care Services, University of Manitoba. All mouse experiments were approved by the University of Manitoba Animal Care Committee in accordance with the regulation of the Canadian Council for Animal Care.

L. major parasites (MHOM/IL/80/Friedlin) were grown in Grace’s insect medium (Life Technologies Life Technologies) supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. For infection, 7-d stationary-phase promastigotes were washed three times in PBS and counted. Mice were infected by injecting 1 × 10⁶ parasites suspended in 50 μl PBS into the right hind footpad. For challenge infections, healed mice (>12 wk postinfection) were injected with 5 × 10⁶ parasites suspended in 50 μl PBS into the left hind footpad. Lesion development and progression were monitored weekly by measuring the diameter of the infected and uninfected footpads with Vernier calipers. Infected WT mice completely resolved their lesions by 12 wk postinfection. To obtain healed CD40 KO mice, we treated infected CD40 KO and IL-12 KO mice intralesionally with 0.3 μg rIL-12 (PeproTech, Rocky Hill, NJ) three times per week for the first 2 wk of infection. This treatment results in complete lesion resolution by 8–10 wk after infection with L. major.

One day before L. major infection, some naive mice were injected with HVEM-Ig or LTβR-Ig fusion proteins (100 μg/mouse) or control-Ig (Human IgG1; Sigma) i.p. (32). To determine whether LIGHT signaling is important at the initiation (priming) and/or maintenance of established immune response, we initiated HVEM-Ig administration 1 d before (as described earlier) or 2 wk post–L. major infection. To investigate the role of LIGHT in secondary (memory) anti-Leishmania immunity, we injected healed WT and CD40 KO mice with HVEM-Ig or LTβR-Ig (100 μg/mouse) i.p. and then challenged them with L. major on the contralateral footpad the next day. To investigate the role of TNF-related activation-induced cytokine (TRANCE) receptor activator of NF-κB (RANK) pathway in secondary anti-Leishmania immunity, we injected healed WT or CD40KO mice with TRANCE-Ig (100 μg/mouse) i.p. and then challenged them with L. major on the contralateral foot the following day. Challenged mice were further given weekly administration of the fusion protein until sacrificed. Mice were monitored for DTH response at 72 h postchallenge and sacrificed 3 wk later to estimate parasite burden.

At various times postinfection, infected mice were sacrificed and the draining lymph nodes (dLNs) were collected and made into single-cell suspensions. Cells were washed, suspended at 4 million/ml in complete medium (DMEM supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin), and plated at 1 ml/well in 24-well tissue culture plates (Falcon; VWR, Edmonton, AB). Cells were stimulated with or without soluble leishmanial Ag (SLA; 50 μg/ml) for 72 h, and the supernatant fluids were collected and stored at −20°C until assayed for cytokines by ELISA. For intracellular cytokine staining, some cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 h, and brefeldin A (10 μg/ml) was added in the last 2 h to enhance intracellular cytokine accumulation. Cells were harvested and stained for cell-surface markers and intracellular cytokines as previously described (33).

IL-4, IL-12, and IFN-γ concentrations in the cell culture supernatant fluids were measured by sandwich ELISA using paired Abs from BD Pharmingen (San Jose, CA) according to manufacturer’s suggested protocols. The sensitivities of the ELISAs were 15, 31, and 15 pg/ml for IL-4, IL-12, and IFN-γ, respectively.

CD11c⁺ cells were enriched from pooled dLNs (three mice per pool) of 3-d L. major–infected WT mice treated with HVEM-Ig, LTβR-Ig, or control Ig, or from spleens of naive WT or CD40 KO mice using Stem Cell EasySep enrichment kit following manufacturer’s instructions. The enriched cells were then stained with allophycocyanin-conjugated anti-CD8α eFlour-450–conjugated anti-CD103 mAbs and then sorted into CD11c⁺CD8α⁺, CD11c⁺CD8α⁻, CD11c⁺CD103⁺, and CD11c⁺CD103⁻ populations using BD FACSAria III (BD Biosciences, Mississauga, ON). The purity of the different cell population was >96% as assessed by flow cytometry.

Bone marrow–derived DCs (BMDCs) were generated from naive WT, HVEM KO, and LTβR KO mice as described previously (34). In brief, bone marrow cells were isolated from the femur and tibia of mice. BMDCs were differentiated in petri dishes in the presence of 20 ng/ml rGM-CSF (Peprotech, Rocky Hill, NJ). Immature DCs were harvested on day 8, and the purity of DCs (i.e., CD11c⁺ cells) was between 85 and 92% as assessed by flow cytometry.

CD4⁺ T cells were purified from single-cell suspensions of spleens from naive or L. major–infected and healed (>12 wk postinfection) WT or CD40 KO mice by negative selection using CD4⁺ T cell enrichment kit (EasySep; Stemcell Technologies, Vancouver, BC) according to the manufacturer’s suggested protocols. To maximize T cell recovery, we first labeled total spleen cells with CFSE dye as previously described (35) before CD4⁺ T cell enrichment. The purity of T cells was >98% as assessed by flow cytometry.

Sorted splenic DCs or BMDCs were cocultured in U-bottom 96-well tissue culture plates with CFSE-labeled CD4⁺ T cells at a DC/T cell ratio of 1:100. The cells were stimulated with or without freeze-thawed L. major Ag (5 × 10⁶ parasite equivalent per well) for 4 d. Proliferation and IFN-γ secretion were then assessed by flow cytometry. In some experiments, the sorted DC subsets from the dLNs of fusion protein–treated and L. major–infected mice were stimulated in vitro with SLA for 3 d and the level of IL-12p40 in the culture supernatant fluid was assessed by ELISA.

Parasite burden in the primary infected or challenged footpads was quantified by limiting dilution analysis as previously described (33).

Student t test was used to compare mean and SEM between two groups. In some experiments, nonparametric one-way ANOVA was used to compare mean and SD of more than two groups. Tukey’s test was used where there was significant difference in ANOVA. Differences were considered significant when p < 0.05.

We showed previously that blockade of LIGHT by HVEM-Ig or LTβR-Ig affects LPS-induced production of IL-12 by BMDCs in vitro (32). To test whether LIGHT blockade affected the subset of DCs that is induced post–L. major infection, we treated naive mice with HVEM-Ig, LTβR-Ig, or control-Ig before infection with L. major and assessed the subset of DCs present in the dLNs directly ex vivo by flow cytometry. Blockade of LIGHT caused significant (p < 0.05–0.01) reduction in the percentages (Fig. 1A, 1C) and absolute numbers (Fig. 1B, 1D) of CD11c⁺CD8α⁺ (Fig. 1A, 1B) and CD11c⁺CD103⁺ (Fig. 1C, 1D) DCs in the dLNs of infected mice. In addition, we found that both CD11c⁺CD8α⁺ and CD11c⁺CD103⁺ DCs were capable of producing IL-12 (Fig. 1E, 1F). However, the CD11c⁺CD8α⁺ DCs subsets were the major population that produced IL-12 post–L. major infection (Fig. 1E, 1F). Interestingly, IL-12 production by CD11c⁺CD8α⁺ cells was significantly (p < 0.01–0.001) reduced post–HVEM-Ig and –LTβR-Ig treatment (Fig. 1E), whereas only LTβR-Ig fusion protein caused significant reduction in IL-12 production in both CD103⁺ and CD103⁻ DC subsets (Fig. 1F). Taken together, these results show that LIGHT affected the numbers and subsets of DCs in the dLNs, as well as their ability to produce IL-12 after infection with L. major.

LIGHT is important for optimal IL-12 production, the development of CD4⁺ Th1 immune response, and resistance to L. major (32). Because IL-12 is required for both priming and maintenance of anti-Leishmania immunity (36–38), we investigated whether LIGHT is required at both the priming and the maintenance stages of anti-Leishmania immunity. Injection of HVEM-Ig (Fig. 2A, 2B) or LTβR-Ig (data not shown) fusion proteins before or starting at 2 wk after L. major infection resulted in significantly (p < 0.001) bigger lesion size and higher parasite burden than in the control-Ig–treated group (Fig. 2A, 2B). This treatment also severely impaired the production of IFN-γ by CD4⁺ T cells after in vitro stimulation with SLA (Fig. 2C–E). However, the effect of LIGHT blockade was more prominent in mice treated with the fusion protein preinfection, suggesting that although this pathway is important for both effective priming and maintenance of anti-Leishmania immunity, it is more important during the initiation phase of the anti-Leishmania immune response.

Next, we wished to determine whether LIGHT is also important for effective secondary (memory) anti-Leishmania immunity. Therefore, we injected HVEM-Ig or LTβR-Ig into healed B6 mice (>16 wk after primary infection), and after 24 h challenged them with L. major. Injection of HVEM-Ig or LTβR-Ig before secondary challenge had no effect on DTH response (Fig. 3A) and rapid control of parasite proliferation (Fig. 3B). Furthermore, whereas injection of HVEM-Ig or LTβR-Ig significantly impaired IL-12 production by dLN cells from infected naive mice (see Figs. 1, 2), it had no effect on the IL-12 production by cells from healed mice after secondary L. major challenge (Fig. 3C). Taken together, these results suggest that although LIGHT is critical for effective primary immunity, it is dispensable during secondary anti-Leishmania immunity.

Although an earlier report showed that CD40 and CD40L interaction is critical for resistance to L. major (39), a later study showed that this interaction may be dispensable for healing low-dose infection (40). Because IL-12 production is indispensable for resistance to L. major, this observation suggests the existence of an alternative (i.e., CD40-CD40L–independent) pathway for IL-12 production during L. major infection. Indeed, the outcome of L. major infection in CD40- and IL-12–deficient mice after treatment with rIL-12 was completely different. Although treatment of both mice with rIL-12 within the first 2 wk of infection initially led to resolution of cutaneous lesion and reduction in parasite burden, lesion recrudescence (indicative of disease reactivation) occurred in IL-12 KO, but not in CD40 KO, mice after cessation of rIL-12 treatment (Fig. 4A, 4B). Furthermore, although rIL-12–treated CD40 KO mice were resistant to secondary L. major challenge, treated IL-12 KO mice were highly susceptible (Fig. 4C). These observations underscore the critical role of CD40–CD40L interaction in IL-12 production and immunity against L. major infection. More importantly, they further confirm the existence of a CD40-CD40L–independent, IL-12–producing pathway for maintenance of secondary immunity in healed CD40 KO mice.

Our findings implicate LIGHT as playing an important role in IL-12 production and optimal resistance to L. major infection (Figs. 1–3) (32). Because healed CD40 KO mice do not reactivate their lesion (unlike IL-12 KO mice) after cessation of rIL-12 treatment, we hypothesized that their durable resistance is due to LIGHT-dependent IL-12 production. We therefore assessed the impact of HVEM-Ig or LTβR-Ig treatment on secondary anti-Leishmania immunity in rIL-12–treated CD40 KO mice. Unlike WT mice, treatment of healed CD40 KO mice with HVEM-Ig or LTβR-Ig led to loss of immunity to secondary L. major challenge as evidenced by significantly (p < 0.01) smaller DTH response and higher parasite burden at the challenged footpads (Fig. 5A, 5B). In addition, dLN cells from healed CD40 KO mice treated with HVEM-Ig or LTβR-Ig produced significantly (p < 0.01–0.001) less IL-12 (Fig. 5C) and IFN-γ (Fig. 5D) compared with the control-Ig–treated group. Furthermore, the level of IL-4 (Fig. 5E) was significantly (p < 0.01) higher in the HVEM-Ig– or LTβR-Ig–treated group compared with the control-Ig–treated group.

A previous report by Padigel et al. (41) showed that in CD40L KO mice, the TRANCE-RANK pathway mediates IL-12 production leading to resistance to L. major. Therefore, we investigated whether the TRANCE-RANK pathway played a role in the maintenance of resistance in IL-12–treated CD40 KO mice infected with L. major. We found that blockade of TRANCE-RANK pathway by treatment with TRANCE-Ig in healed CD40 KO mice had no impact on DTH response and rapid parasite control after secondary L. major challenge (Fig. 6A, 6B). Collectively, these results indicate that in the absence of CD40, LIGHT plays a critical role in IL-12 production, long-term maintenance of immunity, and resistance to secondary L. major challenge.

Next, we wished to determine the relevant DC population that drives CD40-independent but IL-12–dependent maintenance of anti-Leishmania immunity in healed CD40 KO mice. We sorted splenic DCs (CD11c⁺) from WT and CD40 KO mice into highly enriched CD8α⁺ (CD11c⁺CD8α⁺) or CD8α⁻ (CD11c⁺CD8α⁻) and CD103⁺ (CD11c⁺CD103⁺) and CD103⁻ (CD11c⁺CD103⁻) populations, pulsed them with freeze-thawed L. major, and cocultured them with CFSE-labeled CD4⁺ T cells from healed WT mice, and assessed T cell proliferation and IFN-γ production after 4 d. We found that both CD8α⁺ and CD8α⁻ DCs from WT mice induced comparable proliferation and IFN-γ production by CD4⁺ T cells (Fig. 7A, upper panel, and 7B). In contrast, only CD8α⁺ DCs from CD40 KO mice induced appreciable proliferation and IFN-γ production by CD4⁺ T cells from WT mice (Fig. 7A, lower panel, and 7B). Interestingly, CD103⁺ and CD103⁻ DCs from WT and CD40 KO mice induced comparable numbers of proliferating and IFN-γ–producing CD4⁺ T cells in vitro (Fig. 7C, 7D). Taken together, these results suggest that CD8α⁺ DCs are responsible for maintaining CD40-independent CD4⁺ T cell response and immunity in healed CD40 KO mice.

In mice, LIGHT interacts with HVEM and LTβR, two major receptors expressed on APCs including DCs (19, 22, 23). To determine the key receptor involved in LIGHT-mediated IL-12 production and induction of CD4⁺ T cell response, we cocultured L. major–infected BMDCs from WT, HVEM KO, and LTβR KO with CFSE-labeled CD4⁺ T cells from healed WT mice, and assessed cell proliferation and IFN-γ production after 4 d. As shown in Fig. 8A, IL-12 production was significantly (p < 0.001) reduced in cell-culture supernatants of CD4⁺ T cells cocultured with LTβR KO BMDCs compared with those cocultured with WT or HVEM KO BMDCs. Interestingly, the level of IL-12 was significantly (p < 0.01) lower in the LTβR KO compared with the HVEM KO mice (Fig. 8A). In line with this, the percentage of proliferating IFN-γ–producing CD4⁺ T cells in conditions cocultured with LTβR KO BMDCs was significantly (p < 0.01–0.001) lower than those cocultured with WT or HVEM KO BMDCs (Fig. 8B, 8C). Taken together, our data suggest that although binding of LIGHT to HVEM may play some roles, LTβR is the primary receptor involved in LIGHT-mediated IL-12 production, Th1 response, and immunity to L. major.

We previously reported that LIGHT signaling is critical for optimal IL-12 production and CD4⁺ Th1 development in mice, and its blockade either by using HVEM-Ig fusion protein or targeted gene deletion of LIGHT or HVEM results in susceptibility to L. major infection in the otherwise resistant strain of mice (32). However, this study did not directly show the contribution of LIGHT in IL-12 production post–L. major infection or investigate whether LIGHT is important for early and/or sustained IL-12 production in L. major–infected mice. Furthermore, whether LIGHT is required for secondary anti-Leishmania immunity was not investigated. In this study, we corroborate the importance of LIGHT in primary anti-Leishmania immunity. More importantly, we show that LIGHT is required at both the priming and the maintenance stages of primary anti-Leishmania immunity but is completely dispensable during secondary anti-Leishmania immunity in intact (WT) mice with functional CD40–CD40L interaction. However, in the absence of CD40–CD40L interaction (as is seen in CD40 KO mice), blockade of LIGHT led to dramatically impaired DTH and IFN-γ responses and a consequent inability to control parasite proliferation after secondary L. major challenge. We further found that CD8α⁺ DCs are responsible for maintaining this CD40-independent but IL-12–dependent CD4⁺ T cell proliferation and IFN-γ production in L. major–infected CD40 KO mice manipulated to heal their primary infection by rIL-12 treatment. Collectively, this study identifies LIGHT as an important molecule that is critical for maintenance of IL-12–dependent anti-Leishmania immunity in mice.

LIGHT is expressed on activated T cells, monocytes, granulocytes, and immature DCs (19–21) and binds to three receptors: LTβR, HVEM, and in humans, the decoy receptor, DcR3/TR6 (19, 22, 23). HVEM is expressed on resting T cells, NK cells, monocytes, immature DCs, and endothelial cells (22, 24, 25), whereas LTβR is expressed mostly on stromal cells, DCs, and endothelial cells but is absent on lymphocytes (26, 27). The interaction of LIGHT with its receptors including LTβR, HVEM, and in humans, the decoy receptor, DcR3/TR6 (19, 22, 23), modulates several biological processes including cell survival, inflammation, and upregulation of intracellular adhesion molecule-1 leading to tumor eradication (28). In addition, signals transmitted via LIGHT–HVEM interaction have costimulatory effects on T cells, enhancing their proliferation and cytokine production (19, 21). Thus, LIGHT expression enhances cytolytic T lymphocyte–mediated tumor immunity and allograft rejection (21, 29), and its overexpression on T cells results in extensive T cell–proliferative disorders that are characterized by massive polyclonal expansion of CD4⁺ and CD8⁺ T cells (30, 31). Several studies show that the engagement of LIGHT with its ligand plays a crucial role for optimal T cell activation and differentiation into effector cells (21, 22, 42, 43). Thus, blockade of LIGHT–HVEM interaction in vitro inhibits polyclonal T cell proliferation (22, 42), and T cells from LIGHT-deficient mice show impaired effector functions after anti-CD3 stimulation in vitro (44, 45). In addition to having a direct effect on T cells, LIGHT also indirectly influences activation and differentiation, and effector T cell responses by inducing DC maturation and IL-12 production (46, 47). We found that blockade of LIGHT by treatment with HVEM-Ig or LTβR-Ig significantly impairs expression of costimulatory molecules and IL-12 production by total CD11c⁺ cells (data not shown), as well as CD11c⁺CD8α⁺ and CD11c⁺CD103⁺ (Fig. 1) DCs in the dLNs, leading to impairment of Th1 response post–L. major infection. This is in agreement with a recent publication that showed that LIGHT–HVEM interaction is critical for IL-12–, TNF-, and IFN-γ–producing T cells that are responsible for controlling hepatic infection in L. donovani–infected mice (48). Taken together, our results suggest that the interaction of LIGHT with its ligands on APCs (such as DCs) is an important pathway for LIGHT-mediated IL-12 production, T cell activation, and resistance to L. major in mice. Previously published data showed that injection of LTβR agonistic Ab in mice with established L. donovani infection led to protective immune response and parasite clearance, suggesting that LTβR is the key ligand involved in LIGHT-mediated anti-Leishmania immunity (48). Using BMDCs from mice lacking HVEM or LTβR, we corroborate this finding and further show that LIGHT interaction with LTβR was more critical than its interaction with HVEM in mediating a protective immune response because BMDCs from LTβR KO mice induced significantly less proliferation and IFN-γ production in CD4⁺ T cells compared with BMDCs from HVEM KO mice.

IL-12 is a critical cytokine for the development and maintenance of Th1 cells and resistance to L. major (6, 7, 49). Hence we investigated at what stage during Leishmania infection that LIGHT is required for immunity. Treatment of WT mice with HVEM-Ig or LTβR-Ig before (priming/initiation of immunity) or after 2 wk (maintenance of immunity) of infection resulted in significantly bigger lesions and higher parasite burden than in untreated controls (Fig. 2A, 2B). This was associated with severely impaired production of IFN-γ by cells from spleens and dLNs of infected mice (Fig. 2C). Collectively, these observations suggest that akin to rIL-12, LIGHT is required at both the initiation and the maintenance stages of anti-Leishmania immunity. Interestingly, we found that treatment with HVEM-Ig and LTβR-Ig had no effect on IL-12 production, induction of DTH response, and accelerated parasite clearance (Fig. 3). This indicates that although LIGHT contributes to optimal development of primary anti-Leishmania immunity, it has no effect during secondary (memory) immunity to L. major. It is likely that the requirements for activating/recalling memory T cells are different from those needed for differentiation of naive cells into effector cells, with the latter being more dependent on LIGHT-mediated IL-12 production than the former. Indeed, previous reports show that the requirements for activating memory cells are relatively less stringent than those for naive cells, with memory cells requiring lesser costimulation, Ag, TCR-peptide avidity, and cytokines than naive cells (50, 51).

Treatment of infected IL-12–deficient mice with rIL-12 leads to resolution of cutaneous lesion and control of parasite proliferation (37, 38). However, recrudescence occurs after cessation of rIL-12 treatment, suggesting that continuous IL-12 is important for initiation and maintenance of resistance in L. major–infected mice (38). We found that unlike IL-12–deficient mice, treatment of CD40-deficient mice with rIL-12 leads to resistance that is maintained indefinitely even after cessation of recombinant cytokine treatment (Fig. 4). This suggests the existence of a CD40-independent IL-12–producing pathway during the maintenance phase of anti-Leishmania immunity. We found that blockade of LIGHT in CD40-deficient mice that healed their lesion led to impaired DTH response and parasite clearance after secondary L. major challenge that was associated with significantly reduced IFN-γ response. Collectively, these observations show that the maintenance of resistance in L. major–infected, CD40-deficient mice after cessation of rIL-12 treatment is mediated by LIGHT-dependent IL-12 production. Thus, in the absence of CD40 signaling, LIGHT plays a critical role for IL-12 production, a finding that explains the differences in outcome of L. major infection in IL-12– and CD40-deficient mice after cessation of rIL-12 treatment.

A previous report showed that signaling via the TRANCE-RANK pathway could compensate for CD40–CD40L interaction in the production of IL-12 during primary L. major infection (41). However, whether the TRANCE-RANK pathway contributes to IL-12 production during secondary anti-Leishmania immunity is not known. We found that blockade of TRANCE–RANK interaction did not affect IL-12 production and resistance to secondary L. major challenge in CD40-deficient mice, suggesting that this pathway is not responsible for the maintenance of resistance to L. major in CD40-deficient mice after cessation of rIL-12 treatment. Collectively, the data presented in this article suggest that in the absence of CD40, IL-12 production via LIGHT pathway is enough to maintain secondary protective immunity against L. major.

In summary, we have demonstrated that LIGHT is important for DC maturation and IL-12 production after infection with L. major, and disruption of this pathway leads to impairment in CD4⁺ Th1 development and IFN-γ production. Furthermore, we show that although LIGHT is important for the maintenance of anti-Leishmania immunity, it is dispensable for secondary (memory) anti-Leishmania immunity in WT mice. However, in the absence of CD40-CD40L signaling, LIGHT-dependent IL-12 production, primarily via CD8α⁺ DCs, is critical for maintenance anti-Leishmania immunity. Our data corroborate our initial report and further suggest that LIGHT could provide a new therapeutic target for regulation of IL-12 production in vivo and for controlling various CD4⁺ Th1-mediated autoimmune and inflammatory diseases such as arthritis, diabetes, colitis, among others.

We thank the members of Parasite Vaccine Development Laboratory for insightful comments and constructive criticisms.

The authors have no financial conflicts of interest.