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The Journal of Immunology, 2007, 179, 4093-4100
Copyright © 2007 by The American Association of Immunologists, Inc.
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Ligation of B and T Lymphocyte Attenuator Prevents the Genesis of Experimental Cerebral Malaria1

Bernd Lepenies*, Klaus Pfeffer{dagger}, Michelle A. Hurchla{ddagger}, Theresa L. Murphy{ddagger}, Kenneth M. Murphy{ddagger}, Juliane Oetzel*, Bernhard Fleischer* and Thomas Jacobs2,*

* Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; {dagger} Institute of Medical Microbiology, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany; and {ddagger} Department of Pathology and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Plasmodium falciparum malaria remains one of the leading causes of morbidity and mortality, especially in sub-Saharan Africa. The most severe complication of this disease is cerebral malaria (CM),3 often leading to death in humans (1). Besides the high mortality rate, persistent neurocognitive deficits after recovery have become an increasing concern. Infection of susceptible mouse strains with P. berghei ANKA (PbA) is an experimental model of CM that shares characteristics with the human disease (2, 3). Similar to CM in humans, leukocytes are sequestered in brain capillaries in PbA-infected mice developing CM (4, 5, 6). Studies using neutralizing Abs or T cell-deficient mice have clearly demonstrated a role for T cells. Nude mice, SCID, and recombinant Ag (RAG)-deficient mice do not develop CM upon infection with PbA (4, 7, 8, 9). Furthermore, proinflammatory cytokines contribute to the induction of CM in PbA-infected mice (10, 11). We recently found that T cell activation during malaria is accompanied by an increased CTLA-4 expression on CD4+ T cells in humans as well as in rodents (12, 13). In PbA-infected C57BL/6 mice, we found that blockade of CTLA-4 led to enhanced immune pathology triggered by T cells producing proinflammatory cytokines (13, 14). This supports the idea that the rapid induction of CTLA-4 during the blood stage of a PbA infection is a means to counterregulate the strong and polarized activation of the TH1 arm of the immune system to prevent immune pathology.

B and T lymphocyte attenuator (BTLA) is a recently discovered inhibitory receptor on T cells that shares structural and functional similarities with CTLA-4 and PD-1 (15). Recently, the interaction partner of BTLA herpesvirus entry mediator (HVEM) has been identified, which is a member of the TNFR superfamily (16). HVEM is predominantly expressed by resting T cells, monocytes, and immature dendritic cells (DC), but also by endothelial cells (17, 18, 19). BTLA is constitutively expressed by naive CD4+ and CD8+ T cells and is further up-regulated upon T cell activation. It is also present on B cells, macrophages, and bone marrow-derived dendritic cells (20). BTLA cross-linking leads to tyrosine phosphorylation and recruitment of Src homology domain 2-containing protein tyrosine phosphatase 1 and -2 via ITIM/immunoreceptor tyrosine-based switch motif of BTLA (21). In accordance with the role of BTLA as a negative receptor, mice lacking the full-length form of BTLA are hyperresponsive to TCR-mediated activation of T cells (15). In acute allergic airway inflammation, BTLA in combination with PD-1 contributes to the termination of this TH2-mediated immune response, because mice lacking these receptors show prolonged lung inflammation (22). Moreover, it has been demonstrated in an in vivo transplantation model that BTLA is involved in the acceptance of partially MHC-mismatched cardiac allografts (23). A recent study demonstrated a role of the BTLA-HVEM interaction in CD8+ T cell-intrinsic homeostasis and memory cell generation since mice deficient in BTLA or its ligand HVEM have an increased number of memory CD8+ T cells (24).

In the present study, we sought to analyze whether BTLA as a coinhibitory receptor on T and B cells has a functional role in the induction of experimental cerebral malaria in PbA-infected mice. We show here that BTLA is induced in the brain due to infiltration of BTLA-expressing T cells, whereas in spleen BTLA expression is up-regulated on individual T cells after their activation. Treatment of PbA-infected C57BL/6 mice with anti-BTLA mAb (6A6) that blocks BTLA-HVEM interaction but also leads to BTLA ligation in vivo significantly reduced the incidence of CM compared with control mice and led to reduced sequestration of CD4+ and CD8+ T cells in the brains of infected mice. These findings indicate that BTLA-HVEM interactions are functionally involved in T cell regulation during PbA blood-stage malaria and regulate sequestration of T cells in brain capillaries. This study also suggests that BTLA is a potential target for therapeutic interventions in severe malaria.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and parasites

C57BL/6, Tg(TcraTcrb)1100Mjb (OT-1), and Tg(TcraTcrb)425Cbn/J (OT-2) mice were housed in the animal facility of the Bernhard Nocht Institute for Tropical Medicine (Hamburg, Germany). HVEM-deficient mice were generated as described previously (25) and were backcrossed at least five times into the C57BL/6 strain. Genotyping for the HVEM alleles was performed by PCR using the primers HVEM-SP-5' (5'-tccctgaggctgagaggttcc-3') and pJak2 (5'-ctgaagaggagtttacgtccag-3') for the inactivated allele and the primers HVEM-SP-5' and HVEM-SP-3'-wild-type (5'-agaggcagcagggtcagctgg-3') for the wild-type allele. PbA was maintained by alternating cyclic passage of the parasite in Anopheles stephensi mosquitoes and BALB/c mice at the mosquito colony of the Bernhard Nocht Institute for Tropical Medicine. Blood was collected from highly parasitemic mice, and aliquots were stored in liquid nitrogen in a solution of 0.9% NaCl, 4.6% sorbitol, and 35% glycerol. Female C57BL/6 or HVEM–/– mice (5–8 wk old) were infected i.p. with 1 x 106 PbA-infected RBCs. Parasitemia was determined in Giemsa-stained blood smears from tail blood. For application in vivo, mice received 200 µg of anti-BTLA mAb (6A6) or hamster IgG as control Ig i.p. 1 day before infection.

C57BL/6 mice infected with blood stages of PbA have been shown to develop neurological behavioral changes such as ataxia, convulsions, and coma and usually die between days 6 and 8 postinfection (p.i.). Similarly, in our study, 80% of PbA-infected mice that had received only control IgG exhibited signs of cerebral involvement between days 6 and day 8 p.i., with reduced locomotion and marked ataxia. Mice developing CM were euthanized to avoid unnecessary suffering between days 7 and 9 p.i. All experiments were in accordance with the local Animal Ethics Committee Regulations.

Abs, reagents, and flow cytometry

Hybridoma cells that produced anti-BTLA mAb (6A6, Armenian hamster IgG) were routinely maintained in CELLine bioreactors (Integra Biosciences) with IMDM containing 10% FCS and 2 mM L-glutamine. Anti-BTLA mAb 6A6 was purified from supernatants by HiTrap protein G columns (GE Healthcare) using standard protocols. Activity of the mAb was always determined by staining BTLA on splenocytes from C57BL/6 mice with the purified Ab. Quantification of protein was performed using the Bradford assay and verified by SDS-PAGE followed by silver staining. Quantification of endotoxin in the mAb preparation was performed with the Limulus amebocyte lysate kit (QCL-1000; BioWhittaker) to rule out the possibility that observed Ab effects might be influenced by LPS contaminations. Anti-CD3 mAb was purified from the hybridoma cell line 145-2C11. For flow cytometry the following FITC-, PE-, PE-Cy5-, or APC-labeled Abs and appropriate isotype controls from BD PharMingen were used: anti-CD4 (RM4-5), anti-CD19 (1D3), anti-CD25 (7D4), anti-CD45-PerCP (30-F11), anti-CD62L (MEL-14), and anti-CD69 (H1.2F3). Anti-CD8a (CT-CD8a) was obtained from Caltag Laboratories and anti-Foxp3 (FJK-16s) from eBioscience. For analysis of BTLA expression, PE-labeled anti-BTLA (6F7) was used (eBioscience). In each experiment, PE-labeled mouse IgG1, {kappa} (P3) was used as isotype control. Data were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed with the CellQuest program (BD Biosciences).

Quantification of BTLA and HVEM expression using RT-PCR

For expression analysis of BTLA and HVEM, samples were either obtained from mice before onset of CM (day 6 p.i.) or from the group of mice that remained free of cerebral symptoms (days 9 and 12 p.i.). Total RNA was extracted from several organs of uninfected or PbA-infected mice using TriReagent (Molecular Research Center). Contaminating genomic DNA was digested using DNase I (Applied Biosystems). Oligo(dT)-primed cDNA was prepared from 2 µg of RNA using PowerScript Reverse Transcriptase (BD Biosciences) according to the manufacturer’s instructions. Serial dilutions were performed to adjust the amounts of cDNA to equal concentrations. Specific primers for BTLA, HVEM, and beta-actin were designed (BTLA: forward 5'-cctctggagcatcctttgtgagaa-3', reverse 5'-attggtggcatctgggatgtcaga-3'; HVEM: forward 5'-ggttaccatgtgaagcaggtc-3', reverse 5'-ttgactggaaacctgatggtgtt-3'; and beta-actin: forward 5'-gtcgtaccacaggcattgtgatgg-3', reverse 5'-gcaatgcctgggtacatggtgg-3'). One microliter of each cDNA sample was used for subsequent PCR amplification. Band intensities of PCR products were quantified by densitometric analysis using the program Quantity One (Bio-Rad).

Cryosections of brain and staining of brain lymphocytes

C57BL/6 mice were treated with 200 µg of anti-BTLA mAb or control IgG on day –1 and were subsequently infected with 1 x 106 PbA-infected RBCs. On day 6 p.i., mice were sacrificed and brains were removed. Samples were embedded in tissue-freezing medium (Leica), frozen in liquid nitrogen, and stored at –70°C until use. Eight-micrometer-thick frozen sections of brain were prepared and unspecific binding was blocked by incubation with the IgG fraction from Cohn II. Staining was performed with purified hamster anti-mouse ICAM-1 and biotinylated anti-CD31 Ab (BD Pharmingen). Binding was visualized using anti-hamster-tetramethylrhodamine isothiocyanate and streptavidin-Cy5 (Jackson ImmunoResearch Laboratories). Nuclei of cells were stained with 4',6-diamidino-2-phenylindole (Sigma-Aldrich). ICAM-1 expression on brain endothelium was quantified by counting ICAM-1+ vessels per field of view.

For quantification of cell numbers in brain by flow cytometry, brains were removed, homogenized, and RBCs were lysed by addition of ammonium chloride. Staining was performed with anti-CD45-PerCP, anti-CD4-allophycocyanin or anti-CD8-allophycocyanin, and anti-CD62L-PE (BD Pharmingen). In brief, 1 x 107 nucleated cells in brain were analyzed and gated on CD45+ cells. The number of CD4+CD62Llow and CD8+CD62Llow cells in the brain was determined by flow cytometry.

Spleen cell culture and analysis of cytokine production

For cytokine quantification in sera of infected mice, blood was obtained by cardiac puncture after a deep anesthetization with isoflurane. Indirect sandwich ELISAs were performed for the quantification of IL-1beta, IL-6, IL-10, IL-12p40, IL-12p70, IL-18, IFN-{gamma}, and TNF-{alpha}. Ab pairs and cytokine standards were purchased from R&D Systems. For analysis of cytokine production in supernatants of stimulated spleen cells, spleens were removed and RBCs were lysed by addition of ammonium chloride. Single-cell suspensions were cultivated at 2 x 105/well in 96-well plates. Splenocytes from C57BL/6 mice were stimulated with 1 µg/ml anti-CD3, spleen cells from OT-1 with different concentrations of OVA257–264 peptide, and spleen cells from OT-2 cells with OVA323–339 (MWG-Biotech). After 48 h, supernatants were removed and cytokine concentrations were analyzed by ELISA. Indirect sandwich ELISAs were performed for quantification of IL-2, IFN-{gamma}, and IL-10 from supernatants of spleen cells.

Statistical analysis

Statistical analyses were performed with the unpaired Student t test. One-way ANOVA was used to compare parasitemia between different groups of mice. The log rank test was used to test the equality of survival functions across different groups of mice. All statistical analyses were performed with the Prism software (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of BTLA during blood-stage infection with PbA

The blood-stage malaria infection is accompanied by a strong activation of T cells. To verify a potential role of BTLA on the regulation of this T cell response, we first examined the expression of BTLA during blood-stage PbA infection by semiquantitative RT-PCR. BTLA mRNA was markedly induced in several organs on day 9 p.i. (Fig. 1A). Induction was strongest in spleen and kidney, but was also detectable in brain and liver (Fig. 1B). To study whether the induction of BTLA mRNA was accompanied by increased BTLA expression on T cells, we analyzed the kinetics of BTLA expression by flow cytometry. On spleen cells, BTLA was expressed by naive CD4+ and by CD8+ T cells (Fig. 1C). During the course of PbA infection, BTLA expression was up-regulated on both types of T cells (Fig. 1, C and D).


Figure 1
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  		FIGURE 1. Induction of BTLA during PbA infection. A, Total RNA was isolated from whole organs of uninfected C57BL/6 mice (–) or mice infected with PbA-infected RBCs on day 9 p.i. (+). RT-PCR revealed induction of BTLA mRNA in brain, liver, spleen, and kidney. Densitometric quantification of band intensities for BTLA mRNA normalized to the mRNA of the housekeeping gene beta-actin is shown in B. Data are expressed as mean ± SEM. A summary of three independent experiments is shown. C, Splenocytes of C57BL/6 mice were isolated from uninfected mice (solid line) and PbA-infected mice on day 9 p.i. (dashed line), stained with PE-Cy5-labeled anti-CD4 or anti-CD8, and counterstained with BTLA-PE or an appropriate isotype control (dotted line). FACS analysis revealed an increased BTLA expression on day 9 p.i. The time course of BTLA expression by splenic CD4+ T cells ({square}) and CD8+ T cells (Figure 1) during PbA infection is shown in D. Data are expressed as mean ± SEM for each group. A summary of three independent experiments is shown. Statistical significance was tested with the unpaired Student t test (*, p < 0.05; ***, p < 0.0001).

 
Infection of C57BL/6 mice with PbA causes CM in ~80% of infected mice, leading to severe vascular changes in brains with stasis in many capillaries and disruption of the vessel walls with bleeding. Mice that suffer from this pathological event during PbA infection usually die between days 7 and 9 p.i. We found by RT-PCR analysis that BTLA as well as its ligand HVEM were induced in brains of infected mice (Fig. 2A). Furthermore, a semiquantitative analysis of BTLA and HVEM mRNA expression revealed a stronger induction when mice suffered from CM compared with those with uncomplicated malaria (Fig. 2B). We then examined the number of T cells that had migrated into the brain, since it is known that the adhesion of T cells to brain endothelium in synergy with secretion of proinflammatory cytokines contribute to the induction of CM. Indeed, we found strong accumulation of both CD4+BTLA+ and CD8+BTLA+ T cells in the brain during malaria, whereas almost no T cells were detected in the brains of uninfected mice (Fig. 2C). The overall number of CD8+ T cells in the brain of PbA-infected mice exceeded that of CD4+ T cells.


Figure 2
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  		FIGURE 2. Induction of BTLA is due to accumulation of T cells in brain during PbA infection. A, Total RNA was isolated from whole brains of uninfected (not inf.) and PbA-infected C57BL/6 mice with uncomplicated malaria (day 9 p.i., M) or CM (day 8 p.i., CM). B, Densitometric analysis of band intensities (normalized to the mRNA of beta-actin) showed a marked induction of BTLA and HVEM mRNA in brain when mice developed CM. Data are expressed as mean ± SEM. A summary of two independent experiments is shown. C, Total brain homogenates of uninfected or PbA-infected mice were performed. T cells that had infiltrated the brain were stained with anti-CD45-PerCP, anti-CD4-allophycocyanin, or anti-CD8-allophycocyanin and BTLA-PE or an appropriate isotype control. A total number of 5 x 106 cells was recorded. Cells were gated on CD45+ cells. FACS analysis revealed high numbers of CD4+ and CD8+ T cells in brains of infected mice (upper panel) compared with uninfected mice (lower panel). Infiltrating T cells expressed BTLA, as can be seen compared with the isotype control.

 
Effects of anti-BTLA on the incidence of cerebral malaria during PbA infection

Given that BTLA+ T cells accumulate in the brain, we aimed to study whether BTLA as a negative regulator for T cell function may modulate inflammatory processes in the brain. For this purpose, we used an anti-BTLA mAb that has been described to block BTLA-HVEM interactions (23, 26). To further characterize the properties of the anti-BTLA mAb 6A6 on T cells in vitro, we stimulated OT-1 T cells that possess a transgenic TCR recognizing the SIINFEKL peptide bound on MHC class I (H-2Kb) in the presence of different concentrations of anti-BTLA mAb. Plate-bound anti-BTLA caused a decreased IL-2 production after TCR-dependent stimulation (data not shown). This finding indicates that anti-BTLA 6A6 is not only capable of preventing HVEM binding to BTLA, but also leads to a ligation of BTLA, which is accompanied by decreased T cell response. Similar results were obtained for T cells from wild-type C57BL/6 mice after stimulation with anti-CD3 (data not shown).

To study the role of BTLA during PbA infection in vivo, mice received 200 µg of anti-BTLA 6A6 one day before infection. Upon infection with PbA, mice that had been treated with anti-BTLA mAb exhibited a significantly reduced incidence of CM compared with control mice that had received control hamster IgG (3 of 20 vs 21 of 27; p < 0.0001; Fig. 3A). This finding cannot be ascribed to a direct effect of the anti-BTLA treatment on parasitemia because there was no significant difference in the number of parasitized RBCs between 6A6-treated and control mice on day 6 p.i. (Fig. 3B; p > 0.05).


Figure 3
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  		FIGURE 3. Influence of anti-BTLA treatment on the incidence of CM in PbA infection. C57BL/6 mice were treated with 200 µg of anti-BTLA mAb or control IgG on day –1 and were infected with 1 x 106 PbA-infected RBCs. Survival was evaluated using the log rank test (A). Mice were monitored for parasitemia (B). Data are expressed as mean ± SEM for each group. The summary of three independent experiments is shown.

 
To analyze the mechanism by which the decreased incidence of CM upon anti-BTLA treatment was mediated, we quantified the number of T cells that accumulated in the brain by flow cytometry. For this purpose, we stained CD45+CD4+ and CD45+CD8+ lymphocytes in the brain and also determined the fraction of activated CD62Llow T cells. Upon anti-BTLA treatment, we found a decreased number of CD45+ cells in the brains of mice compared with mice treated with control IgG (Fig. 4A, left panel). The triple staining revealed that CD45+CD8+CD62Llow T cells as well as CD45+CD4+CD62Llow T cells were reduced in the brains of mice on day 6 p.i. after treatment with anti-BTLA (Fig. 4A, right panel, and data not shown). Quantitative analysis revealed a 50% reduction for CD8+ T cells as well as CD4+ T cells in the brains of infected mice (Fig. 4B). To study whether the BTLA ligation in vivo also altered the proinflammatory immune response during PbA infection, we quantified cytokines in sera of mice that had either received anti-BTLA mAb or control IgG. Indeed, mice that were treated with anti-BTLA mAb 1 day before infection exhibited lower serum levels of IL-18 (546 ± 211 pg/ml vs 1183 ± 186 pg/ml, p = 0.024) and IFN-{gamma} (29 ± 29 pg/ml vs 338 ± 189 pg/ml, p = 0.042; Fig. 4C), indicating that the BTLA ligation altered the quality of the ongoing immune response during PbA infection. The concentration of IL-6 in sera of the anti-BTLA-treated group was also reduced compared with control mice although values did not reach statistical significance (Fig. 4C). Regarding other cytokines measured in sera, we found either no significant difference (IL-12p40) or concentrations were at or below the limit of detection (IL-1beta, IL-10, IL-12p70, TNF-{alpha}; data not shown).


Figure 4
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  		FIGURE 4. Reduced infiltration of T cells into the brain of PbA-infected mice upon anti-BTLA treatment. C57BL/6 mice were treated with 200 µg of anti-BTLA mAb or control IgG on day –1 and were infected with 1 x 106 PbA-infected RBCs. On day 6 p.i., cells in brain were stained with anti-CD45-PerCP, anti-CD4-allophycocyanin, or anti-CD8-allophycocyanin and anti-CD62L-PE. A, Representative dot plots are shown for cells in brain of anti-BTLA-treated mice (upper panel) and mice that had received control Ig (lower panel). In brief, 1 x 107 cells were analyzed and cells were gated on CD45+ cells in the brain. FACS analysis showed an increased frequency of cells that were CD8+CD62Llow in the control group. B, Numbers of CD8+CD45+ and CD4+CD45+ T cells per 1 x 107 nucleated cells in brain are shown. Cells were isolated from uninfected mice (Figure 4), anti-BTLA-treated mice (•), and mice treated with control Ig ({circ}) before PbA infection. Data are expressed as mean ± SEM. One of two representative experiments is shown. Statistical significance was tested using the unpaired Student t test (*, p < 0.05; **, p < 0.01). C, Decreased concentrations of proinflammatory cytokines in sera of PbA-infected mice upon anti-BTLA treatment. On day 6 p.i., IL-18, IFN-{gamma}, and IL-6 were measured in sera of C57BL/6 mice that had been treated with anti-BTLA mAb (n = 7) or control IgG (n = 6) on day –1 (mean of duplicates for each mouse). Data are expressed as mean ± SEM. Statistical significance was tested using the unpaired Student t test. The dashed line in each diagram represents cytokine levels in sera of uninfected mice.

 
A possible mechanism by which the reduced sequestration of T cells in the brains of anti-BTLA-treated mice during PbA infection might be mediated is an altered expression of adhesion molecules on brain endothelium. To assess the relevance of adhesion molecules for protection against CM in anti-BTLA-treated mice, we stained ICAM-1 on frozen sections of the brain. ICAM-1 was not expressed on brain endothelium of uninfected mice, whereas CD31 as a constitutively expressed endothelial marker was detectable (Fig. 5A). ICAM-1 expression was induced in the brains of either anti-BTLA-treated or control mice during PbA infection (Fig. 5A). When we compared the expression levels of ICAM-1, we found no difference between anti-BTLA-treated and control mice, suggesting that BTLA ligation has no direct effect on the expression of adhesion molecules in brain (Fig. 5B). However, we verified the reduced number of CD8+ T cells that were sequestered in the brains of mice that had received anti-BTLA mAb (data not shown).


Figure 5
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  		FIGURE 5. No alteration in the expression of ICAM-1 on brain endothelium upon anti-BTLA treatment. C57BL/6 mice were treated with 200 of µg anti-BTLA mAb (upper panel) or control IgG (middle panel) on day –1 and were infected with 1 x 106 PbA-infected RBCs. On day 6 p.i., frozen sections of the brain were stained for CD31 (yellow), ICAM-1 (red), and 4'-diamidino-2-phenylindole (DAPI; blue). Bottom panel, A staining of brain sections from an uninfected C57BL/6 mouse. One representative experiment of three independent experiments is shown in A. B, ICAM-1 expression was quantified by counting ICAM-1+ vessels per field of view. Data are expressed as mean ± SEM. A summary of three experiments is shown.

 
Since anti-BTLA prevents the genesis of CM in mice when administered 1 day before infection, we next analyzed whether treatment of mice with anti-BTLA was successful at later time points (Table I). Application of anti-BTLA 6A6 on day 2 p.i. also resulted in protection against CM in a high number of mice (6 of 8 vs 1 of 8, p = 0.006, compared with control IgG). However, when treatment started on day 4 p.i. or during early symptoms of CM (reduced locomotion), no protective effect of the anti-BTLA mAb was observed. This finding indicates that anti-BTLA has to be present during priming and expansion of T cells to exert the described effects.


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  		Table I. Incidence of CM during PbA infection is reduced by anti-BTLA treatment until day 2 p.i.

 
Although it has been shown previously that anti-BTLA mAb 6A6 blocks BTLA-HVEM interactions and is also capable of ligating BTLA, we investigated the possibility that BTLA+ cells might be depleted by this mAb in vivo. To this end, we stimulated splenocytes ex vivo from mice that had been treated with anti-BTLA or control Ig and were subsequently infected. Stimulation of spleen cells from infected mice with anti-CD3 Ab revealed no significant differences in IFN-{gamma} as well as IL-10 production between both groups of mice, indicating that T cells are still able to produce cytokines upon stimulation (data not shown). Furthermore, FACS analysis revealed no alteration in cellular composition in the spleen upon treatment of mice with anti-BTLA before PbA infection and no significant alterations in the expression of activation markers CD25, CD62L, and CD69 were observed (Fig. 6, A and B). Next, we investigated whether anti-BTLA treatment influenced the frequency of regulatory T cells (Treg) in spleen. However, no difference in the frequency of CD4+CD25+Foxp3+ cells was found, indicating that a direct effect of anti-BTLA on Treg is rather unlikely (Fig. 6, A and B). To rule out depletion of a subpopulation of BTLA+ spleen cells, we stained splenic T cells from infected mice that had received the anti-BTLA mAb 6A6 by using an anti-BTLA mAb (6F7) that recognizes a different epitope of BTLA and observed no alterations in the frequency of BTLA+ cells compared with control mice (data not shown). These findings demonstrate that the anti-BTLA mAb 6A6 does not induce a systemic immune suppression or a depletion of activated cells that up-regulate BTLA. Thus, it can be clearly excluded that the observed effects on the incidence of CM might be caused by a depletion of BTLA-expressing CD4+ or CD8+ T cells.


Figure 6
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  		FIGURE 6. No alteration of cellular composition and activation markers in the spleen during PbA infection upon anti-BTLA treatment. C57BL/6 mice were treated with 200 µg of anti-BTLA mAb (upper panel) or control IgG (middle panel) on day –1 and were infected with 1 x 106 PbA-infected RBCs. A, On day 6 p.i., spleen cells were stained with anti-CD4-allophycocyanin and counterstained either with anti-CD8-PE, anti-CD62L-PE, anti-CD69-PE, or anti-CD25-FITC (uninfected control in the bottom row). Influence of the anti-BTLA treatment on the frequency of Treg was determined by triple staining of splenocytes with anti-CD4-allophycocyanin, anti-CD25-FITC, and anti-Foxp3-PE. One characteristic experiment of three is shown. Absolute numbers of cell subpopulations in spleen are shown in B. FACS analysis revealed no significant alterations of the cellular composition in the spleen or expression of activation markers. Data are expressed as mean ± SEM. A summary of three independent experiments is shown. Dashed lines represent cell numbers in the spleen of uninfected mice.

 
To confirm that the observed effects are due to a ligation of BTLA by anti-BTLA mAb 6A6 as seen in vitro, we infected HVEM–/– mice. PbA-infected HVEM–/– mice exhibited a similar parasitemia and incidence of CM as wild-type control mice (Fig. 7, A and B). This finding demonstrates that the costimulatory interaction of HVEM-LIGHT is dispensable for the development of CM in this model and that ligation of BTLA by endogenous HVEM is not sufficient to attenuate the ongoing inflammation. Interestingly, treatment of PbA-infected HVEM–/– mice with anti-BTLA led to a reduced incidence of CM compared with HVEM–/– and wild-type control mice (Fig. 7A). This was accompanied by decreased sequestration of CD4+ as well as CD8+ T cells in the brain (Fig. 7C) to an extent that was comparable to the effect observed in wild-type mice (Fig. 4B). These data clearly indicate that not a blockade of the HVEM-BTLA interaction by the anti-BTLA mAb 6A6 causes the decreased incidence of CM, but ligation of BTLA by this Ab.


Figure 7
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  		FIGURE 7. Infection of HVEM–/– mice with PbA. HVEM–/– mice, wild-type control mice, and HVEM–/– mice that had received 200 µg of anti-BTLA mAb 1 day before infection were infected with 2 x 106 PbA-infected RBCs. Survival was evaluated using the log rank test (A). Mice were monitored for parasitemia (B). Data are expressed as mean ± SEM for each group. The summary of two independent experiments is shown. Statistical significance was tested with ANOVA. C, HVEM–/– mice were treated with 200 µg of anti-BTLA mAb (•) or control IgG ({circ}) on day –1 and were infected with 2 x 106 PbA-infected RBCs. On day 6 p.i., brains were isolated, homogenized, and RBCs were lysed by addition of ammonium chloride. Cells were stained with anti-CD45-PerCP, anti-CD4-allophycocyanin, or anti-CD8-allophycocyanin and CD62L-PE. Numbers of CD8+CD45+ and CD4+CD45+ T cells per 1 x 107 nucleated cells in brain are shown. Data are expressed as mean ± SEM. Statistical significance was tested using the two-tailed unpaired t test (*, p < 0.05; **, p < 0.01).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we used PbA infection of C57BL/6 mice to examine whether the coinhibitory receptor BTLA has a functional role in the development and/or control of CM. Numerous studies have illustrated that activated T cells that are sequestered in the brain contribute to CM (8, 27). Treatment of mice with anti-LFA-1 mAb inhibiting LFA-1/ICAM-1 interaction and thereby adhesion of T cells to the endothelium prevents mice from developing CM (28, 29). Moreover, the development of CM in PbA-infected C57BL/6 mice is strongly dependent on the production of proinflammatory cytokines during the course of infection (11, 30). We have shown in earlier studies that during the blood stage of PbA infection a strong induction of CTLA-4, another negative regulator expressed on activated T cells, occurs (13). Using the PbA model, we found an exacerbation of the disease upon CTLA-4 blockade that was caused by an increased number of activated T cells producing proinflammatory cytokines. Thus, CTLA-4 is involved in down-regulation of effector functions of T cells during the course of malaria infection. In contrast, in Plasmodium yoelii NL infection, a model that is characterized by a more balanced immune response accompanied by the production of IL-4, IL-10, TGF-beta, and IFN-{gamma}, we observed a reduced peak parasitemia and earlier parasite clearance after anti-CTLA-4 treatment (31). These data indicate that T cells contribute to protection, but may also enhance pathology during a blood-stage malaria infection of mice, which indicates that a T cell response has to be tightly regulated.

Comparable to the data obtained for CTLA-4 expression, we observed a strong induction of BTLA expression on splenocytes during PbA blood-stage infection, although in contrast to CTLA-4 BTLA is constitutively expressed by naive T cells. This finding is consistent with already published data showing that BTLA is induced upon TCR-mediated stimulation of T cells in vitro (20). Recently, it has been shown in vivo that BTLA contributes to the termination of allergic airway inflammation because BTLA-deficient mice exhibit a prolonged infiltration of lymphocytes and eosinophils in bronchoalveolar lavage fluid (22). During blood-stage PbA infection, we found that BTLA expression on splenic CD4+ as well as CD8+ T cells increased during the course of disease. BTLA induction was also observed in brain, liver, and kidney in which activated T cells migrate upon infection. It has previously been shown that HVEM expression is not restricted to immune cells but also expressed on endothelial cells, which would be a prerequisite for modulation of T cells in the periphery (19). Indeed, we found that HVEM was up-regulated in the brain during infection. Interestingly, mice that suffered from CM displayed a higher HVEM level than mice with uncomplicated (noncerebral) malaria. Given the observed kinetics of HVEM induction and infiltration of BTLA+ T cells in the brain, the HVEM-BTLA interaction might contribute to prevent overwhelming immune pathology by limiting the release of proinflammatory cytokines.

To study whether BTLA induction is functionally involved in T cell regulation during murine malaria infection, we used an anti-BTLA Ab (6A6) in vivo that has been described to block HVEM-BTLA interaction in vitro (26). Using TCR-transgenic T cells, we found that anti-BTLA mAb 6A6 inhibited T cell function by BTLA ligation. In the PbA infection model, we found that application of anti-BTLA caused a significant reduction in the incidence of CM in comparison to control mice. This was accompanied by decreased levels of proinflammatory cytokines in sera of anti-BTLA-treated mice. The effect of the anti-BTLA mAb on the genesis of CM was not mediated by an altered expression of adhesion molecules on brain endothelium since no alteration in ICAM-1 expression was detected. To exclude the possibility that the anti-BTLA mAb might deplete BTLA+ T cells, we examined the cellular composition in spleen and the frequency of activation markers on splenocytes. We found no differences which indicate that this Ab does not alter total cell numbers or the frequency of cell populations in spleen. This finding was corroborated by staining BTLA on T cells from infected mice that were treated with anti-BTLA 6A6 with a different anti-BTLA Ab. Because mice that were treated with anti-BTLA 6A6 or control Ig displayed a similar number of BTLA+ T cells upon infection, a depletion of T cells can be excluded. Furthermore, ex vivo-stimulated splenocytes, isolated from infected mice that had received anti-BTLA or control Ig, showed no significant alterations in cytokine production, which argues against a general suppression of T cells. However, treatment with anti-BTLA was accompanied by a strong reduction of T cell sequestration in the brain.

It has been shown that HVEM is not only expressed by resting T cells, NK cells, monocytes, and immature dendritic cells, but also by endothelial cells (19). To exclude that BTLA might act as an adhesion molecule in the brain that causes accumulation of T cells by interaction with HVEM on brain endothelial cells, we used infection of HVEM-deficient mice. It is important to notice that HVEM does not only bind to BTLA, but also to LIGHT, a member of the TNF superfamily, and thus transmits a costimulatory signal to the LIGHT-expressing cells (32). HVEM-deficient mice exhibited a similar parasitemia and incidence of CM as wild-type mice accompanied by sequestration of T cells in the brain. This finding demonstrates that the costimulatory activity of HVEM/LIGHT is neither involved in the induction of CM nor in parasite clearance. Moreover, this finding excludes that the HVEM-BTLA interaction contributes directly to the adhesion of T cells to the endothelium as it is known for LFA-1/ICAM-1 interactions in malaria (8, 27). However, we found that anti-BTLA treatment of HVEM-deficient mice led to a decreased sequestration of T cells in the brain during infection. This suggests that ligation of BTLA, but not the blockade of HVEM-BTLA interaction prevents sequestration of T cells in the brain and the induction of CM. In a recent study, a role for BTLA in sustaining cell survival during chronic allostimulation has been proposed (33). In a model of acute graft-versus-host disease (GVHD), BTLA–/– donor T cells failed to sustain GVHD, suggesting a previously unexpected function of BTLA in promoting immune responses. Anti-BTLA mAb 6A6 was shown to inhibit the long-term survival of BTLA+/+ donor lymphocytes and thereby ameliorated GVHD. This effect was explained by a blockade of BTLA in vivo leading to a loss of GVHD-promoting donor cells. In our study, we show that during PbA infection of mice anti-BTLA acts as an agonistic Ab in vivo since anti-BTLA-treatment also leads to a reduced incidence of CM in PbA-infected HVEM–/– mice. However, we cannot exclude that other yet unidentified receptors for BTLA might have an influence on BTLA signaling.

In conclusion, the present study provides evidence that BTLA+ T cells are involved in the development of CM in a murine malaria model using PbA infection. Ligation of BTLA in vivo is accompanied by a reduced sequestration of T cells in the brain, decreased levels of proinflammatory cytokines in serum, and prevents the genesis of CM without inducing general immune suppression. Thus, interference with the BTLA-HVEM interaction might be a means to modulate T effector functions in inflamed organs during infectious diseases.


   Acknowledgment
 
We thank Iris Gaworski for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Deutsche Forschungsgemeinschaft Grant JA 1451 (to T. J.). Back

2 Address correspondence and reprint requests to Dr. Thomas Jacobs, Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, Hamburg, Germany. E-mail address: tjacobs{at}bni-hamburg.de Back

3 Abbreviations used in this paper: CM, cerebral malaria; PbA, Plasmodium berghei ANKA; BTLA, B and T lymphocyte attenuator; HVEM, herpesvirus entry mediator; p.i., postinfection; Treg, regulatory T cell; GVHD, graft-versus-host. Back

Received for publication December 12, 2006. Accepted for publication July 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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