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The Journal of Immunology, 2007, 179: 3178-3186.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Complement-Dependent Enhancement of CD8+ T Cell Immunity to Lymphocytic Choriomeningitis Virus Infection in Decay-Accelerating Factor-Deficient Mice1

Chongyun Fang*, Takashi Miwa*, Hao Shen{dagger} and Wen-Chao Song2,*

* Institute for Translational Medicine and Therapeutics and Department of Pharmacology, and {dagger} Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Decay-accelerating factor (DAF, CD55) is a GPI-anchored membrane protein that regulates complement activation on autologous cells. In addition to protecting host tissues from complement attack, DAF has been shown to inhibit CD4+ T cell immunity in the setting of model Ag immunization. However, whether DAF regulates natural T cell immune response during pathogenic infection is not known. We describe in this study a striking regulatory effect of DAF on the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) infection. Compared with wild-type mice, DAF knockout (Daf-1–/–) mice had markedly increased expansion in the spleen of total and viral Ag-specific CD8+ T cells after acute or chronic LCMV infection. Splenocytes from LCMV-infected Daf-1–/– mice also displayed significantly higher killing activity than cells from wild-type mice toward viral Ag-loaded target cells, and Daf-1–/– mice cleared LCMV more efficiently. Importantly, deletion of the complement protein C3 or the receptor for the anaphylatoxin C5a (C5aR) from Daf-1–/– mice reversed the enhanced CD8+ T cell immunity phenotype. These results demonstrate that DAF is an important regulator of CD8+ T cell immunity in viral infection and that it fulfills this role by acting as a complement inhibitor to prevent virus-triggered complement activation and C5aR signaling. This mode of action of DAF contrasts with that of CD59 in viral infection and suggests that GPI-anchored membrane complement inhibitors can regulate T cell immunity to viral infection via either a complement-dependent or -independent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The protein decay-accelerating factor (DAF)3 (CD55) is a ubiquitously expressed GPI-anchored membrane protein that restricts complement activation on autologous cells (1, 2). DAF inhibits complement activation by preventing the formation and accelerating the decay of C3 and C5 convertases of both the classical and alternative pathways (1, 2). Aside from complement regulation, DAF has also been implicated in T cell biology via a number of potential mechanisms (3, 4, 5). As a GPI-anchored protein, DAF has been found to localize to the lipid-raft fraction of the cell membrane where it may complex with nonreceptor tyrosine kinases and participate in signal transduction during T cell activation (4). DAF has also been identified as a ligand for an activation-associated, G protein-coupled receptor-like, lymphocyte Ag CD97 (6, 7, 8, 9). A recent study has demonstrated that co-engagement of CD3 and DAF, using either cross-linking mAbs or its natural ligand CD97, results in enhanced proliferation of human peripheral blood CD4+ T cells (10). In both cases, the role of DAF in T cell activation appeared to be independent of its activity as a complement regulator.

A role for DAF in cellular immunity is also supported by studies of DAF knockout (Daf-1–/–) mice (11, 12, 13). In previous studies, we found that DAF deficiency significantly exacerbated systemic autoimmunity in MRL/lpr mice (12), and C57BL/6 Daf-1–/– mice immunized with two model Ags, OVA and myelin oligodendrocyte glycoprotein (MOG), exhibited markedly enhanced CD4+ T cell recall response (13). Furthermore, Daf-1–/– mice immunized with MOG developed much more severe symptoms of experimental autoimmune encephalomyelitis, a typical CD4+ T cell-mediated inflammatory condition of the CNS (13). One of the limitations of the immunization experiments with OVA and MOG was the use of CFA and the fact that the procedure primarily elicited CD4+ T cell responses. It is not known whether the regulatory effect of DAF on T cell immunity is restricted to CD4+ T cells and whether it can be observed in settings of natural T cell immune reactions during pathogenic infections. In the present study, we have addressed these questions by examining the CD8+ T cell response of wild-type (WT) and Daf-1–/– mice to lymphocytic choriomeningitis virus (LCMV) infection. We describe in this study that both the primary and memory CD8+ T cell responses to acute or chronic LCMV infection was dramatically enhanced in Daf-1–/– mice. Significantly, we found that the phenotype of enhanced CD8+ T cell immunity in Daf-1–/– mice was completely dependent on C3 or the receptor for the anaphylatoxin C5a. These results support the conclusion that DAF is a significant modulator of CD8+ T cell immunity in the setting of natural microbial infection and that it plays such a role by functioning as a complement regulator. Our data further highlight the connection between complement and T cell immunity and support a critical role of C5aR in the interaction between the two systems.


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

Daf-1–/– mice deficient in the Daf-1 gene, the murine homolog of human DAF, were generated by gene targeting as previously described (11). They were backcrossed onto the C57BL/6 background for 10 generations. Six time-backcrossed C57BL/6-C3–/– mice were obtained from The Jackson Laboratory, and were backcrossed in house for an additional five generations. A breeder pair of C5a receptor knockout (C5aR–/–) mice on the C57BL/6 background was provided by Dr. J. Lambris (University of Pennsylvania, Philadelphia, PA) (14). C3–/– or C5aR–/– mice were cross-bred with Daf-1–/– mice to derive Daf-1–/–/C3–/– or Daf-1–/–/C5aR–/– mice, respectively. Male mice ages 8–12 wk were used in this study. Gender- and age-matched WT C57BL/6 mice were obtained from The Jackson Laboratory. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Reagents

Allophycocyanin-conjugated rat anti-mouse IFN-{gamma} (clone XMG1.2), PE- or FITC-conjugated rat anti-mouse CD8a (clone 53-6.7), PerCp-conjugated rat anti-mouse CD4 (clone RM4-5), allophycocyanin-conjugated rat anti-mouse CD44 (clone IM-7), FITC-conjugated rat anti-mouse CD62 ligand (CD62L, clone MEL-14), purified rat anti-mouse Fc receptor (clone 2.4G2), GolgiStop and Cytofix/Cytoperm kit were from BD Pharmingen. DMEM, L-glutamine, HEPES, PBS, nonessential amino acids, sodium pyruvate, 2-ME, and penicillin-streptomycin were from Invitrogen Life Technologies. LCMV gp33–41 peptide was synthesized by Sigma-Aldrich. FBS was from HyClone Laboratories. [3H]thymidine was from Amersham Biosciences. EL-4 cell line was from American Type Culture Collection.

Infection of mice with LCMV

LCMV-Armstrong and LCMV-clone 13 strains were propagated in vitro, and viral titers were determined by plaque assays as previously described (15). For acute LCMV infection, 2 x 105 PFU of LCMV-Armstrong were administered to mice by i.p. injection. Mice were sacrificed at day 6, 7, or 8 to collect the spleens for analysis of primary immune response or 2 mo later for memory immune response. For chronic LCMV infection, 2 x 106 PFU LCMV-clone 13 were administered to mice by i.v. injection through the tail vein. Mice were bled at day 8, 15, 30, and 45 to obtain PBMC for T cell analysis, and to obtain serum for monitoring virus clearance. Mice infected with clone 13 were sacrificed at day 50 postinfection for terminal analysis of splenocytes. Experiments were also performed to test the function of the memory immune response to LCMV infection. For these experiments, mice were first infected with 2 x 105 PFU LCMV-Armstrong (i.p.) and then challenged 5 mo later with 2 x 106 PFU LCMV-clone 13 (i.v.). Mice were sacrificed at different time points, and the titers of LCMV-clone 13 virus in their spleens were determined by plaque assays as previously described (15).

Preparation of splenocytes and PBMC

Spleens were first cut into small pieces and meshed with the blunt end of a plastic syringe in a petri dish on ice. Single-cell suspension was prepared by passing the spleen homogenate through a 70-µm cell strainer (Falcon) and cell pellets were collected after centrifugation at 600 x g for 7 min. Contaminating RBC were lysed by resuspending the cell pellets in ACK buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA (pH 7.3)) for 3 min and then washing twice in PBS. To prepare PBMC, 100 µl of blood was collected from each mouse by retro-orbital bleeding using sodium citrate as an anticoagulant. RBC were removed by ACK lysis followed by washing with PBS. Numbers of splenocytes and PBMCs were determined on a Beckman Coulter counter.

Analysis of cell surface markers

A total of 1 x 106 splenocytes or 5 x 105 PBMC in 30 µl of staining buffer (PBS containing 1% BSA and 0.1% NaN3) were first treated with the anti-Fc receptor mAb 2.4G2 (Fc Block) on ice for 15 min to reduce nonspecific staining. The cells were then stained with various combinations of Abs for 30 min at 4°C in the dark. After washing twice with staining buffer, cells were fixed in 1% paraformaldehyde and analyzed by FACS on a FACSCalibur instrument using the CellQuest software (BD Biosciences). Data were analyzed with the FlowJo software (Tree Star).

In vitro Ag restimulation of splenocytes and intracellular staining for IFN-{gamma}

Splenocytes were adjusted to 1 x 107 cells/ml in complete medium (DMEM containing 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids, 50 µM 2-ME, 1 mM sodium pyruvate, and 100 U/ml penicillin-streptomycin). A total of 100 µl of the cell suspension was seeded into each well of a 96-well U-bottom plate. Another 100 µl of complete medium containing GolgiStop (used at 1/750 dilution) and the LCMV gp33–41 peptide (final concentration, 0.01 µM) was added to each well. After 5 h of culture, cells were harvested and were stained sequentially for surface CD8a and intracellular IFN-{gamma}. Surface staining was performed as described and intracellular staining was performed with the Cytofix/Cytoperm kit. The frequency of IFN-{gamma}-producing cells in gated CD8+ T cells was determined by FACS analysis.

Quantitation of LCMV-specific CD8+ T cells using MHC-tetramer

Allophycocyanin-conjugated gp33–41/Db tetramer was prepared in-house and used at 1/500 dilution as previously described (16). Splenocytes were first stained with gp33–41/Db tetramer for 30 min at room temperature in the dark, followed by staining with FITC-conjugated rat anti-mouse CD8a at 4°C for another 30 min. After washing in PBS, cells were fixed in 1% paraformaldehyde and analyzed by FACS.

CTL assay

JAM test was used to determine the CTL activity of splenocytes from LCMV-infected mice. This method is based on the principle of DNA fragmentation after cell death (17). EL-4 cells (a mouse lymphoma cell line, C57BL/6 background) were used as target cells. Cells were cultured in complete DMEM, and exponentially growing cells were treated with the gp33–41 peptide (final concentration, 0.01 µM) and [3H]thymidine (final concentration, 5 µCi/ml) for 5 h at 37°C. After extensive washing in DMEM, the gp33–41 peptide-pulsed and [3H]thymidine-pulsed EL-4 cells were mixed at different ratios with splenocytes from LCMV-Armstrong infected WT or Daf-1–/– mice (day 7 after LCMV infection) in 96-well U-bottom plates. After cell mixing, plates were centrifuged at 200 x g for 5 min and incubated for 5 h at 37°C in a 5% CO2 incubator. Cells were harvested onto glass fiber filters using a cell harvester (Tomtec) and the specific CTL activity was determined by scintillation counting according to the following formula: percentage of specific lysis = ((cpm from spontaneous release wells – cpm from experimental wells)/cpm from spontaneous release wells) x 100, where the average cpm from experimental wells (wells containing both target cells and effectors) and the average cpm from spontaneous release wells (wells containing target cells only) (17) are used.

Statistical analysis

Data were expressed as mean ± SEM. Groups were compared by two-tailed, unpaired Student’s t test and the significance was defined at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Daf-1–/– mice had markedly increased total and Ag-specific CD8+ T cell expansion upon acute LCMV infection

To determine whether DAF regulates CD8+ T cell immunity, we infected groups of WT and Daf-1–/– mice with LCMV-Armstrong, a virus strain that causes acute infection and a strong CD8+ T cell response in the mouse (15, 18, 19). On day 6, 7, or 8 postinfection, mice were sacrificed, and the percentage and total number of CD4+ and CD8+ T cells in their spleens were determined by FACS. As shown in Fig. 1, A and B, Daf-1–/– mouse spleens were found to contain a significantly higher percentage of CD8+ T cells than WT mouse spleens at day 7 postinfection (30.40 ± 1.60% vs 20.48 ± 1.45%, n = 4 mice per group, p < 0.005). The total number of CD8+ T cells in Daf-1–/– mouse spleens was also greatly increased (32.80 ± 2.76 x 106/spleen vs 12.50 ± 1.64 x 106/spleen, p < 0.001) (Fig. 1C). Similar increases in CD8+ T cell expansion in Daf-1–/– mice were also observed on days 6 and 8 postinfection (Fig. 1D). Because there was no difference between naive (noninfected) WT and Daf-1–/– mice in their splenic CD8+ T cell frequency or total number (data not shown and Ref. 20), these results suggested that DAF deficiency either promoted Ag-specific CD8+ T cell expansion or impaired the depletion of nonspecific CD8+ T cells in response to acute LCMV infection. We observed no significant difference in the frequency of splenic CD4+ T cells between LCMV-infected WT and Daf-1–/– mice (Fig. 1A). However, in three of four independent experiments, the total number of splenic CD4+ T cells was significantly greater in Daf-1–/– mice than in WT mice (data not shown).


Figure 1
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  		FIGURE 1. Increased CD8+ T cell expansion after acute LCMV infection in Daf-1–/– mice. WT and Daf-1–/– mice (n = 4 for each group) were infected with LCMV-Armstrong. At indicated time points postinfection, the percentage and total number of CD4+ and CD8+ T cells in their spleens were determined by FACS analysis. A, Representative FACS analysis showing increased frequency (percentage of cells shown) of CD8+ but not CD4+ T cells in the spleens of Daf-1–/– mice. B, Scatter plot of splenic CD8+ T cell frequencies in WT and Daf-1–/– mice (n = 4). C, Scatter plot of splenic CD8+ T cell numbers in WT and Daf-1–/– mice (n = 4). D, Time course analysis of CD8+ T cell frequency in the spleens of WT and Daf-1–/– mice (day 6, 7, and 8). **, p < 0.001. E, Representative FACS analysis of gated splenic CD8+ T cells showing a higher percentage (each quadrant) of CD8+ T cells had an activated phenotype (CD44high/CD62Llow) in Daf-1–/– mice. F, Scatter plot of the percentage of CD44high/CD62Llow cells among gated splenic CD8+ T cells in WT and Daf-1–/– mice (n = 4). Except in D, all data are from day 7 postinfection. Data are representative of four independent experiments.

 
Studies have shown that in the early phase of LCMV infection, nonspecific T cells are deleted to make space for the expansion of LCMV-specific T cells (21, 22, 23). The activated, Ag-specific T cells can be distinguished by their up-regulation of CD44 and down-regulation of CD62L (CD44high/CD62Llow) (23, 24). To determine whether the increased CD8+ T cell number in Daf-1–/– mice was due to LCMV-driven expansion of Ag-specific T cells or ineffective depletion of nonspecific T cells, we analyzed the expression of CD44 and CD62L on gated CD8+ T cells. As shown in Fig. 1, E and F, the frequency of CD44high/CD62Llow cells among gated CD8+ T cells was significantly higher in Daf-1–/– mice than that in WT mice (80.83 ± 0.75% vs 72.30 ± 1.33%, n = 4 mice, p < 0.0014). This implied that the much larger CD8+ T cell population in Daf-1–/– mouse spleens most likely resulted from LCMV-driven expansion of Ag-specific CD8+ T cells rather than inefficient depletion of nonspecific CD8+ T cells.

To further characterize the highly expanded CD8+ T cells in Daf-1–/– mice, we analyzed the number of splenic CD8+ T cells that were specific for the LCMV antigenic gp33–41 peptide by staining total splenocytes with the gp33–41/Db tetramer (16). Fig. 2, A–C, shows that the percentage and total number of gp33–41/Db tetramer-positive CD8+ T cells in Daf-1–/– mouse spleens were significantly higher than that in the WT mouse spleens (percentage: 2.02 ± 0.15% vs 1.30 ± 0.10%; total number: 21.84 ± 2.39 x 105/spleen vs 7.76 ± 0.43 x 105/spleen; n = 4 mice per group, p < 0.01 for both measurements). Separately, we stimulated splenocytes from LCMV-infected WT and Daf-1–/– mice with gp33–41 and analyzed the number of IFN-{gamma}-secreting CD8+ T cells after intracellular staining of IFN-{gamma}. Fig. 2, E–G, shows that Daf-1–/– mouse spleens were again found to contain a significantly higher percentage and total number of IFN-{gamma}-producing CD8+ T cells than WT mouse spleens (percentage: 4.44 ± 0.41% vs 2.50 ± 0.29%; total number: 48.23 ± 6.18 x 105/spleen vs 14.99 ± 1.57 x 105/spleen; n = 4 mice, p < 0.01 for both measurements). These data indicated that Daf-1–/– mice had increased Ag-specific CD8+ T cell expansion upon acute LCMV infection. Notably, the relative percentage of gp33–41/Db tetramer-positive (Fig. 2D) or IFN-{gamma}-producing cells (Fig. 2H) among gated CD8+ T cells showed a trend of increase in Daf-1–/– mice but did not differ significantly from that of WT mice, suggesting that DAF deficiency caused expansion of gp33–41-specific as well as other epitope-specific CD8+ T cells.


Figure 2
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  		FIGURE 2. Daf-1–/– mouse spleens contained more LCMV-specific CD8+ T cells. Splenocytes from mice infected with LCMV-Armstrong (day 7 after infection) were analyzed directly by gp33–41/Db tetramer staining or by IFN-{gamma} intracellular staining after in vitro restimulation with the gp33–41 peptide. A, Representative FACS analysis showing that Daf-1–/– mouse spleens contained more gp33–41/Db tetramer-positive CD8+ T cells than WT mouse spleens. Scatter plots of the frequency (B) and number (C) of gp33–41/Db tetramer-positive CD8+ T cells (per spleen) in WT and Daf-1–/– mice (n = 4 mice each group). D, Scatter plot of the frequency of gp33–41/Db tetramer-positive cells among gated CD8+ T cells. E, Representative FACS analysis showing that Daf-1–/– mouse spleens contained more IFN-{gamma}-secreting CD8+ T cells than WT mouse spleens. Scatter plots of the frequency (F) and number (G) of IFN-{gamma}-secreting CD8+ T cells (per spleen) in WT and Daf-1–/– mice (n = 4). H, Scatter plot of the frequency of IFN-{gamma}-secreting cells among gated CD8+ T cells.

 
Daf-1–/– mouse splenocytes exhibited higher total CTL activity and contained lower titers of LCMV

To evaluate the functional significance of enhanced Ag-specific CD8+ T cell immunity in Daf-1–/– mice, we compared the total CTL activities of WT and Daf-1–/– mouse splenocytes at day 7 after LCMV-Armstrong infection. For this experiment, EL-4 cells pulsed with the viral antigenic peptide gp33–41 were used as target cells (25). Fig. 3A shows that the total CTL activity of Daf-1–/– mouse splenocytes was significantly higher than that of WT mouse splenocytes at three different E:T ratios. This most likely reflected an increased number of gp33–41-specific CD8+ T cells in Daf-1–/– mice rather than higher CTL activity of individual Daf-1–/– CD8+ T cells. Indeed, in a separate assay wherein we normalized the effector cell number in splenocytes based on the frequency of gp33–41/Db tetramer-positive CD8+ T cells, we observed no difference in total CTL activity between WT and Daf-1–/– mice (data not shown). In a parallel experiment, we investigated the antiviral activity of CD8+ T cells in vivo by determining the LCMV titers in WT and Daf-1–/– mouse spleens at day 3, 5, and 7 post infection with LCMV-Armstrong. As expected, we observed that the virus titers in both groups of mice decreased exponentially between days 3 and 7, indicating that the virus was being cleared by the host (Fig. 3B). Notably, we found that the viral titer in the spleen of Daf-1–/– mice was significantly lower than that in the spleen of WT mice at days 5 and 7, suggesting that Daf-1–/– mice eliminated LCMV more efficiently than WT mice. These data correlated well with the increased CD8+ T cell number in Daf-1–/– mice as described and suggested that the more vigorously expanded CD8+ T cells in the mutant mice were functionally relevant.


Figure 3
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  		FIGURE 3. Daf-1–/– mouse splenocytes had higher killing activity and lower virus titers. WT and Daf-1–/– mice (n = 4 per group) were infected with LCMV-Armstrong. A, On day 7 postinfection, splenocytes (effectors) were assayed for killing activity toward gp33–41-pulsed EL-4 cells (targets). Daf-1–/– mouse splenocytes showed higher killing activities at all three E:T ratios. Each curve represents splenocytes from a single mouse. B, In separate experiments, virus titers in the mouse spleens were determined by plaque-assays on days 3, 5, and 7 postinfection. Results are representative of two independent experiments each. **, p < 0.01 between WT and Daf-1–/– groups.

 
Enhanced CD8+ T cell response to chronic LCMV infection in Daf-1–/– mice

The kinetics of viral clearance and immune response to chronic LCMV infection is significantly different from that of acute LCMV infection (26). Chronic LCMV infection is characterized by a high virus load in multiple tissues for 2–3 mo (15). Although the infection is eventually under control in most tissues, the virus is not completely eliminated from the host (15). The induction of CD8+ T cell response in chronic LCMV infection also differs in that, although CD4+ T cells are not necessary for an acute CD8+ T cell response, they are indispensable for CD8+ T cell responses in chronic LCMV infection (27, 28). To examine the CD8+ T cell response of Daf-1–/– mice during chronic LCMV infection, we infected groups of WT and Daf-1–/– mice with LCMV clone 13, a more virulent LCMV strain that causes chronic infection in mice (15). We then monitored their CD8+ T cell response by analyzing PBMC (day 8, 15, 30, and 45) or splenocytes (day 50, terminal) for the frequency of CD8+ or gp33–41/Db tetramer-positive T cells. As shown in Fig. 4, A and B, we found that the percentage of CD8+ or gp33–41/Db tetramer-positive T cells in PBMC of Daf-1–/– mice was significantly higher than the percentage found in WT mice at all time points examined. Notably, increased total and gp33–41-specific CD8+ T cells in Daf-1–/– mice correlated with lower blood viral titers in these mice at latter but not early time points after infection (Fig. 4C). When splenocytes were restimulated at day 50 with gp33–41, a higher percentage (Fig. 4, D and E) and total number (Fig. 4F) of Daf-1–/– mouse splenocytes secreted IFN-{gamma} as assessed by intracellular staining (percentage: 0.32 ± 0.01% vs 0.13 ± 0.01%; total number: 2.3 ± 1.26 x 105/spleen vs 0.80 ± 0.28 x 105/spleen, n = 4 mice per group, p < 0.001 for both measurements). These data demonstrated that Daf-1–/– mice could mount a persistently stronger CD8+ T cell response to chronic LCMV infection.


Figure 4
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  		FIGURE 4. Daf-1–/– mice had increased CD8+ T cell response to chronic LCMV infection. WT and Daf-1–/– mice (n = 4 for each group) were infected with LCMV-clone 13 and their PBMC or splenocytes were examined at different time points. A, Percentage of CD8+ T cells in PBMCs at various time points after infection. B, Percentage of gp33–41/Db tetramer-positive CD8+ T cells in PBMCs. C, Blood LCMV titers at various time points after infection. D, Representative FACS analysis showing that, after restimulation in vitro with gp33–41, splenocytes from LCMV-clone 13-infected Daf-1–/– mice (day 50) had more IFN-{gamma}-secreting CD8+ T cells than that of WT mice. Scatter plots of the frequency (D) and number (E) of IFN-{gamma}-secreting CD8+ T cells in the spleens of WT and Daf-1–/– mice. Results are representative of two independent experiments. *, p < 0.05; **, p < 0.01.

 
Daf-1–/– mice developed more Ag-specific memory CD8+ T cells

In the case of acute LCMV infection, most of the activated T cells are eliminated by activation-induced cell death after resolution of virus infection (16). Only ~5% of the these cells will survive and develop into memory T cells, which can give rise to a quicker and more vigorous recall response upon reencounter with the same Ag. To determine whether DAF deficiency also influences the development of memory CD8+ T cells, groups of WT and Daf-1–/– mice were infected with LCMV-Armstrong and examined 2 mo later. Mice were sacrificed and splenocytes were analyzed for the frequency of gp33–41/Db tetramer-positive cells directly or of IFN-{gamma}-producing CD8+ T cells after gp33–41 restimulation. Although there was no difference in the spleen size or total number of splenocytes between the two groups of mice, Daf-1–/– mice were found to contain significantly more gp33–41/Db tetramer-positive T cells in their spleens than WT mice (percentage: 0.95 ± 0.08% vs 0.52 ± 0.03%; total number: 4.58 ± 0.27 x 105/spleen vs 2.75 ± 0.35 x 105/spleen; n = 5 mice per group, p < 0.005 for both measurements) (Fig. 5, A–C). Similarly, in response to gp33–41 peptide restimulation, Daf-1–/– mouse spleens contained significantly more IFN-{gamma}-secreting CD8+ T cells than WT mouse spleens (percentage: 1.09 ± 0.05% vs 0.70 ± 0.03%; total number: 5.18 ± 0.43 x 105/spleen vs 2.51 ± 0.27 x 105/spleen; n = 5 mice, p < 0.001 for both measurements) (Fig. 5, D–F). These data indicated that more memory CD8+ T cells had developed in Daf-1–/– mouse spleens after acute LCMV infection.


Figure 5
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  		FIGURE 5. Daf-1–/– mice developed more memory CD8+ T cells after acute LCMV infection. WT and Daf-1–/– mice (n = 5) were infected with LCMV-Armstrong and LCMV-specific memory CD8+ T cells were examined 2 mo later by gp33–41/Db tetramer and intracellular IFN-{gamma} staining. A, Representative FACS analysis showing that Daf-1–/– mouse spleens contained more gp33–41/Db tetramer-positive CD8+ T cells than WT mouse spleens. Scatter plots of the frequency (B) and number (C) of splenocytes that were gp33–41/Db tetramer-positive CD8+ T cells in the spleens of WT and Daf-1–/– mice (n = 5). D, Representative FACS analysis showing that, after restimulation with gp33–41, more IFN-{gamma}-secreting CD8+ T cells were detected in Daf-1–/– mouse splenocytes than in WT mouse splenocytes. Scatter plots of the frequency (E) and number (F) of splenocytes that were IFN-{gamma}-secreting CD8+ T cells in WT and Daf-1–/– mice (n = 5).

 
Memory T cells can be divided into two subpopulations according to the level of CD62L expression: CD62Llow effector memory T cells and CD62Lhigh central memory T cells (29, 30). It has been thought that CD62Lhigh central memory T cells are derived from CD62Llow effector memory T cells and that the former are more potent in controlling secondary viral infection (30, 31). To further characterize the memory CD8+ T cell phenotype in Daf-1–/– mice, we analyzed CD62L expression on gp33–41/Db tetramer-positive CD8+ T cells in Daf-1–/– and WT mice 3 mo after LCMV-Armstrong infection. Interestingly, we found that the percentage of CD62Llow cells among gated gp33–41/Db tetramer-positive CD8+ T cells was significantly higher in Daf-1–/– mice than the percentage found in WT mice (Fig. 6, A and B). This result suggested that the increased memory CD8+ T cells in Daf-1–/– mice were biased toward an effector memory CD8+ T cell phenotype.


Figure 6
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  		FIGURE 6. Phenotypic and functional assessment of LCMV-specific memory CD8+ T cells. WT and Daf-1–/– mice (n = 5) were infected with LCMV-Armstrong and their LCMV-specific CD8+ T cells were assessed after 3 or 5 mo. A, Representative FACS analysis of splenocytes at 3 mo postinfection showing that a higher percentage (shown in each quadrant) of CD62Llow cells were detected in Daf-1–/– mice than in WT mice among gated gp33–41/Db tetramer-positive CD8+ T cells. B, Scatter plot of the ratio of effector memory CD8+ T cells (CD62Llow) to central memory CD8+ T cells (CD62Lhigh) in WT and Daf-1–/– mice (n = 5). C, Scatter plot showing that the average titer of LCMV-clone 13 virus in the spleens of Daf-1–/– mice was lower than that in WT mice (n = 5). Mice were re-infected with LCMV-clone 13 five months after LCMV-Armstrong infection and spleens were examined for LCMV-clone 13 virus load at day 5 after re-infection.

 
To evaluate the functional significance of increased memory CD8+ T cells in Daf-1–/– mice in vivo, we infected groups of Daf-1–/– and WT mice with LCMV-Armstrong, and after 5 mo, rechallenged these groups with the more virulent LCMV-clone 13 strain. Five days after LCMV-clone 13 infection, mice were sacrificed and their spleens were collected to determine the virus titer by plaque assay. Fig. 6C shows that the virus titer in Daf-1–/– mouse spleens was significantly lower than that in WT mouse spleens, suggesting that Daf-1–/– mice mounted a more effective anti-LCMV recall response.

Enhanced CD8+ T cell response to LCMV infection in Daf-1–/– mice is C3- and C5aR-dependent

To explore the mechanism of enhanced CD8+ T cell immunity to LCMV infection in Daf-1–/– mice, we investigated the consequences of C3 or C5aR gene deficiency in Daf-1–/– mice. Daf-1–/– mice were crossed with C3–/– and C5aR–/– mice, respectively, to generate Daf-1–/–/C3–/– and Daf-1–/–/C5aR–/– mice. Comparison of the total and Ag-specific CD8+ T cell expansions upon acute LCMV-Armstrong infection in WT, Daf-1–/–, Daf-1–/–/C3–/–, and Daf-1–/–/C5aR–/– mice revealed that the enhanced CD8+ T cell immunity phenotype in Daf-1–/– mice was completely rescued by either C3 or C5aR deficiency (Fig. 7). This result indicated that enhanced CD8+ T cell immunity to LCMV infection in Daf-1–/– mice was dependent on complement activation and C5aR signaling.


Figure 7
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  		FIGURE 7. Enhanced CD8+ T cell responses in Daf-1–/– mice were complement- and C5aR-dependent. WT, Daf-1–/–, Daf-1–/–/C3–/–, and Daf-1–/–/C5aR–/– mice (n = 4 for each group) were infected with LCMV-Armstrong. On day 7 postinfection, splenocytes were collected and were either analyzed by FACS directly for CD8+ and gp33/Db tetramer-positive T cells or were stimulated with gp33–41 and then analyzed for IFN-{gamma}-secreting CD8+ T cells. A, Scatter plot of CD8+ T cell frequencies in splenocytes of the four groups of mice. B, Scatter plot of the frequencies of gp33/Db tetramer-positive CD8+ T cells in splenocytes of the four groups of mice. C, Scatter plot of the frequencies of IFN-{gamma}-secreting CD8+ T cells in splenocytes of the four groups of mice. In each case, there was significant difference between WT and Daf-1–/– mice but no difference between WT and Daf-1–/–/C3–/– or Daf-1–/–/C5aR–/– mice. NS, p > 0.05; **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have demonstrated in this study that mice deficient in the GPI-anchored membrane complement regulator DAF mounted a markedly more vigorous CD8+ T cell response to LCMV infection. This finding extends our previous observation in Daf-1–/– mice of enhanced CD4+ T cell recall response to model Ag immunization (13) and shows that DAF could regulate both CD4+ and CD8+ T cell immunity. It also shows that such a regulatory effect of DAF on T cell immunity could be manifested during a natural immune response to viral infection as well as in the setting of adjuvant-based single Ag immunization (13). We further demonstrated that the enhanced CD8+ T cell response in Daf-1–/– mice had functional significance in that these mice had lower splenic virus titers at days 5 and 7 after LCMV-Armstrong infection and that their splenocytes exhibited higher total CTL activities. Because there was no difference between WT and Daf-1–/– mice in their virus titers at day 3 postinfection (Fig. 3B), the lower virus load in Daf-1–/– mice at the latter time points must have resulted from accelerated clearance of the virus rather than altered susceptibility to LCMV infection. Similarly, in the chronic LCMV infection model increased CD8+ T cell expansion in Daf-1–/– mice correlated with significantly diminished virus titer at latter time points (Fig. 4C). The higher CTL activity demonstrated by Daf-1–/– mouse splenocytes most likely reflected the increased number of virus-specific CD8+ T cells and not enhanced CTL activity of individual Daf-1–/– CD8+ T cells.

It is notable that the phenotype of increased T cell immunity in LCMV-infected Daf-1–/– mice was more pronounced with CD8+ T cells than with CD4+ T cells. This difference may be related to the nature of LCMV-elicited T cell response as the virus is known to drive primarily a CD8+ T cell response in the mouse (32, 33, 34, 35). Nevertheless, this result contrasted with the specific inhibition of CD4+ T cell immunity by CD59, another GPI-anchored membrane complement regulator, in mice infected with recombinant vaccinia virus (36). A further important difference between the inhibitory functions of DAF and CD59 in T cell immunity is that the activity of CD59 was shown to be complement-independent (36), whereas we have demonstrated that enhanced CD8+ T cell immunity in LCMV-infected Daf-1–/– mice was dependent on C3 and C5aR. Thus, GPI-anchored membrane complement regulators could influence T cell immunity to viral infection via at least two different mechanisms.

The complement-dependent nature of the Daf-1–/– mouse phenotype in LCMV infection is consistent with our previous finding that C3 and C5 were necessary for Daf-1–/– mice to develop a CD4+ T cell recall hyperresponse after OVA or MOG immunization (13). It also agrees with previous studies showing that C3 was required for optimal T cell expansion in response to viral infection in WT mice (37, 38). Suresh et al. (37) have shown earlier that C3 but not CR1/CR2 deficiency caused epitope-specific impairment in CD8+ T cell expansion after LCMV infection in mice of two genetic backgrounds. On the 129/B6 background, NP396–404-specific CD8+ T cells in WT mice were ~2-fold more than that in C3–/– mice, whereas on the C57BL/6 background gp33–41-specific CD8+ T cells were twice as many in WT mice than in C3-deficient mice (37). In a model of influenza virus infection, Kopf et al. (38) demonstrated that C3 deficiency resulted in delayed viral clearance, reduced priming of CD4+ Th cells and CD8+ CTLs, and severely impaired recruitment of virus-specific T cells into the lungs. Consistent with our demonstration of an essential role for C5aR in the Daf-1–/– mouse phenotype, blocking the interaction of C5a-C5aR by a peptide antagonist effectively inhibited the expansion of CD8+ T cells specific for the immunodominant NP366–374 peptide of influenza virus (39). Taken together, these data suggested that complement augmented antiviral T cell immunity via a C5aR-dependent, CR1/CR2-independent mechanism and that in the absence of DAF, this adjuvant effect of complement on T cell immunity was amplified, presumably by increased C5a generation.

Several questions arise from our hypothesis and remain to be addressed experimentally. First, does the LCMV cause more complement activation in Daf-1–/– mice than in WT mice and, if so, does it occur in the fluid phase (plasma) or on specific cell types? Second, how and on what cells does the C5a-C5aR interaction promote T cell immunity? Although we have not attempted to measure and compare complement activation in LCMV-infected WT and Daf-1–/– mice, complement is known to be activated by West Nile virus in vivo in the mouse (40, 41, 42). Furthermore, we have recently found that LPS, a TLR4 ligand and alternative pathway complement activator from Gram-negative bacteria, caused significantly increased systemic complement activation in Daf-1–/– mice than in WT mice (43). It is therefore quite possible that LCMV similarly induced a higher degree of complement activation in Daf-1–/– mice, either systemically or locally on cells it infects. In the case of LPS-treated Daf-1–/– mice, increased complement activation synergized with LPS-mediated TLR4 signaling in a C5aR-dependent manner, resulting in greatly elevated production of inflammatory cytokines including IL-6 and TNF-{alpha} (43). By analogy to LPS-stimulated events, it may be speculated that a similar synergistic interaction between complement and TLR occurred in LCMV-infected Daf-1–/– mice, which promoted dendritic cell maturation and/or T cell priming through elevated inflammatory cytokine production and adhesion/costimulatory molecule expression. Relevant to this hypothesis, previous work by others has shown that LCMV-driven innate and CD8+ T cell immunity in mice was dependent on TLR2 and the obligatory TLR signaling adaptor molecule MyD88 (44).

In summary, we have shown a marked C5aR-dependent enhancement in anti-LCMV CD8+ T cell immunity in Daf-1–/– mice. This finding further highlights the connection between complement and the adaptive immune system and reaffirms the role of DAF as a negative inhibitor of T cell immunity in vivo. The knowledge that complement and its membrane regulators such as DAF mediate primary and memory T cell immunity may be exploited therapeutically in the treatment of human immune disorders and in the development of effective vaccines for cancer and infectious diseases.


   Acknowledgments
 
We thank Drs. John Lambris and Craig Gerard for C5aR–/– breeder mice, and Dr. E. John Wherry (Wistar Institute, Philadelphia, PA) for advice on LCMV plaque assays.


   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 Grants AI-49344, AI-44970, and AI-62388 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Wen-Chao Song, Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, 1254 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: Song{at}spirit.gcrc.upenn.edu Back

3 Abbreviations used in this paper: DAF, decay-accelerating factor; LCMV, lymphocytic choriomeningitis virus; CD62L, CD62 ligand; MOG, myelin oligodendrocyte glycoprotein; WT, wild type. Back

Received for publication June 19, 2007. Accepted for publication June 21, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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