The neurotropic coronavirus JHM strain of mouse hepatitis virus persists in oligodendroglia despite the presence of virus-specific CD8 T cells. Expression of programmed death 1 (PD-1) and B7-H1 were studied during acute and persistent infection to examine whether this negative regulatory mechanism contributes to CNS viral persistence. The majority of CNS-infiltrating CD8 T cells expressed PD-1, with the highest levels on virus-specific CD8 T cells. Moreover, despite control of infectious virus, CD8 T cells within the CNS of persistently infected mice maintained high PD-1 expression. Analysis of virus-susceptible target cells in vivo revealed that B7-H1 expression was regulated in a cell type-dependent manner. Oligodendroglia and microglia up-regulated B7-H1 following infection; however, although B7-H1 expression on oligodendroglia was prominent and sustained, it was significantly reduced and transient on microglia. Infection of mice deficient in the IFN-γ or IFN-α/β receptor demonstrated that B7-H1 expression on oligodendroglia is predominantly regulated by IFN-γ. Ab blockade of B7-H1 on oligodendroglia in vitro enhanced IFN-γ secretion by virus-specific CD8 T cells. More efficient virus control within the CNS of B7-H1-deficient mice confirmed inhibition of CD8 T cell function in vivo. Nevertheless, the absence of B7-H1 significantly increased morbidity without altering demyelination. These data are the first to demonstrate glia cell type-dependent B7-H1 regulation in vivo, resulting in adverse effects on antiviral CD8 T cell function. However, the beneficial role of PD-1:B7-H1 interactions in limiting morbidity highlights the need to evaluate tissue-specific intervention strategies.

Several features of the CNS make it a challenging environment for the immune response to efficiently clear virus. These obstacles include the blood-brain barrier, a specialization of the vasculature in the CNS that limits leukocyte entry (1). In addition, the absence of classical lymphoid drainage and restricted Ag presentation by CNS resident cells hinders induction, recruitment, and retention of adaptive immune cells. Furthermore, in contrast to peripheral infections, several target cells of CNS infections are terminally differentiated, nonrenewable cells which have evolved mechanisms to avoid extensive immune-mediated damage. However, the same mechanisms protecting CNS resident cells from extensive immune-mediated pathology also favor establishment of viral persistence.

A variety of inhibitory molecules and their ligands on T cells and infected cells contribute to T cell dysfunction, thereby fostering viral persistence. Among these are the programmed death 1 (PD-1)5 receptor and its ligand B7-H1, also known as PD-L1, which belong to the B7:CD28 family (2). PD-1 is inducibly expressed on T cells, B cells (3), monocytes (4), and NK cells (5). B7-H1 is constitutively expressed on T cells, B cells, macrophages, and dendritic cells and is further up-regulated after stimulation (6, 7). B7-H1 is also expressed on a variety of nonhematopoietic cell types including vascular endothelial cells (8), epithelial cells (9), and cells of the nervous system (10, 11, 12, 13). PD-1:B7-H1 interactions trigger a noneffective T cell response, referred to as “T cell exhaustion,” which is characterized by impaired proliferation, cytolysis, and cytokine production (8, 9, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Exhausted T cells were initially described in chronic lymphocytic choriomeningitis virus-infected mice (24) and similar dysfunction of T cells has been reported during HIV (19, 20)-, hepatitis B virus (25)-, hepatitis C virus (15, 26, 27)-, and simian immunodeficiency virus (28)-persistent infections. Similar to pathogens exploiting the PD-1:B7-H1 pathway to establish persistence, expression of B7-H1 on tumors may also serve as an immune evasion strategy (29, 30).

The role of inhibitory molecules in nervous system infections has not been explored extensively. Following HSV infection, neurons express Qa-1b, the major ligand for the NK inhibitory receptor CD94/NKG2A expressed by infiltrating HSV-specific CD8 T cells (31). CD94/NKG2-Qa-1b interactions suppress the cytolytic activity of CD8 T cells during HSV latency (31), implicating a regulatory mechanism to protect irreplaceable neurons at the cost of establishing viral latency. Furthermore, B7-H1 expression on neurons prevents viral control of rabies virus, resulting in enhanced CNS viral load and mortality (10). To explore a role for the PD-1:B7-H1 pathway in modulating CD8 T cell-mediated activity toward glia cells within the CNS, regulation of these molecules was assessed in mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV). JHMV primarily infects astrocytes, microglia, and oligodendroglia and induces acute encephalomyelitis and immune-mediated demyelination (32, 33, 34). Although perforin-mediated CD8 T cell cytolysis is the primary mechanism that controls viral replication in astrocytes and microglia (35), viral replication in oligodendroglia is controlled by IFN-γ (36, 37, 38). However, despite an effective immune response in the CNS, oligodendroglia are the targets of persistent infection (37). The failure to clear virus from oligodendroglia is not due to the absence of Ag presentation, as oligodendroglia up-regulate MHC class I- and Ag-processing components, albeit delayed compared with microglia (39). The observation that viral persistence in oligodendroglia is associated with persisting but noneffective CD8 T cells in the CNS (40, 41) suggests that T cell effector function may be dampened by inhibitory ligands.

This report demonstrates that CNS-infiltrating CD8 T cells express elevated PD-l levels which increase further during persistence. Prominent expression of B7-H1 on oligodendroglia, but not microglia, suggests cell type-dependent regulation and differential engagement of CD8 T cells by these cell types in vivo. Blockade of B7-H1 during oligodendrocyte-mediated CD8 T cell stimulation indeed enhanced IFN-γ production in vitro. The dampening effects of B7-H1 in vivo were confirmed by more rapid virus control in B7-H1-deficient mice. These data are the first to demonstrate a significant contribution of B7-H1 in promoting viral persistence in resident CNS cells, while protecting from immune-mediated damage.

C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). Transgenic mice expressing GFP via the oligodendroglia-specific proteolipid protein promoter on the C57BL/6 background were used to analyze oligodendroglia (39, 42). IFN-γ-deficient mice (IFN-γ−/−), IFN-α/β receptor-deficient mice (IFNAR−/−), and B7-H1-deficient mice (B7-H1−/−) on the C57BL/6 background were previously described (37, 43, 44). Mice expressing GFP under control of the glial fibrillary acidic protein promoter (45) on the BALB/c background were used to analyze astrocytes. All mice were bred at an accredited animal facility at the Cleveland Clinic Lerner Research Institute or the University of Southern California. Mice were infected at 6–7 wk of age by intracranial injection with 250 PFU of the J.2.2v-1 mAb variant of JHMV (46). Animals were scored for clinical signs of disease with: 0, healthy; 1, ruffled fur and hunched back; 2, hind limb paralysis or inability to turn to upright position; 3, complete hind limb paralysis and wasting; and 4, moribund or dead. JHMV-specific memory CD8 T cells were elicited by i.p. immunization of 6- to 7-wk-old BALB/c mice with 5 × 106 PFU of JHMV. All procedures were conducted in accordance with federal guidelines under animal protocols approved by the respective institutional animal care and use committees.

For isolation of glial cells, spinals cords (n = 3–5) were finely minced with a razor blade and digested with 2 ml of 0.25% trypsin in PBS for 30 min at 37°C. Trypsin activity was terminated by addition of 4 ml of RPMI 1640 supplemented with 25 mM HEPES and 20% newborn calf serum followed by centrifugation. Cells were washed with RPMI 1640 supplemented with 25 mM HEPES and 1% FCS, resuspended in RPMI 1640, and adjusted to 30% Percoll (Pharmacia). Cell suspensions were underlayed with 1 ml of 70% Percoll, centrifuged at 800 × g for 30 min at 4°C, and cells were recovered from the 30–70% interface. Cells were washed once and resuspended in FACS buffer (PBS with 0.5% BSA). For analysis of infiltrating cells, CNS cells were isolated as described previously (41). Briefly, spinal cords (n = 3–4) in PBS were disrupted in ice-cold Tenbroeck glass tissue homogenizers (Thomas Scientific). Cells were recovered by centrifugation for 7 min at 400 × g and purified for flow cytometric analysis using Percoll gradients as described above. Single cells from the cervical lymph nodes (CLN) and spleen were prepared from identical animals as previously described (41).

Expression of cell surface markers was determined by staining cells with FITC-, PE-, PerCP-, or allophycocyanin-conjugated mAb specific for CD45 (30-F11), CD8 (53-6.7), CD80 (16-10A1; BD Biosciences), MHC class I (28-14-8), PD-1 (RMP1-30), B7-H1 (MIH5), PD-L2 (TY25; eBioscience), and F4/80 (CI:A3-1; Serotec). Isotype control mAb for B7-H1 and MHC class I were rat IgG2a (BD Biosciences) and mouse IgG2a (eBioscience), respectively. Virus-specific CD8 T cells were identified using H2-Db/S510 MHC class I tetramers (Beckman Coulter) as described previously (41). Oligodendroglia from proteolipid protein-GFP mice were identified as CD45GFP+ cells while microglia were identified as CD45lowGFP. Oligodendroglia from IFN-γ−/− and IFNAR−/− were identified by the surface expression of O4, an oligodendrocyte-specific marker (47), using purified anti-O4 mAb followed by anti-IgM conjugated with allophycocyanin (BD Biosciences). Before staining, cells were incubated with mouse serum and rat anti-mouse FcγIII/II mAb (2.4G2; BD Biosciences) for 20 min on ice to minimize nonspecific binding. Cells were then incubated with Ab for 30 min on ice, washed twice with FACS buffer, and analyzed on a FACSCalibur flow cytometer (BD Biosciences) using FlowJo 7.1 software (Tree Star).

The N19 oligodendroglia cell line which displays biochemical and morphological characteristics of oligodendroglia (48) was provided by Dr. A. Campagnoni (University of California, Los Angeles School of Medicine, Los Angeles, CA). N19 cells were grown at 34°C in DMEM/F12 with 10% FCS and G418 antibiotic (130 μg/ml; Sigma-Aldrich). Surface markers were analyzed following treatment with either IFN-γ (5 ng/ml; BD Biosciences) or IFN-β (3 ng/ml; PBL Biomedical Laboratories) for 24 h. For B7-H1 inhibition assays, confluent monolayers were treated with IFN-γ (5 ng/ml) for 24 h, irradiated (3000 rad), and incubated with the immunodominant Ld-restricted viral nucleocapsid peptide at a final concentration of 1 μM at 34°C (41). Cells were then washed three times and used to stimulate splenic CD8 T cells from JHMV-immunized animals either in the presence (10 μg/ml) or absence of anti-B7-H1 mAb (MIH6). Specific binding of MIH6 to its ligand as well as blocking capacity has been previously demonstrated (7, 13, 49). Cultures were incubated for 96 h at 37°C and supernatants were collected at consecutive time points to assess IFN-γ production by ELISA as previously described (50). Student’s t test was used to compare the mean ± SEM for each group. Statistics were assessed using GraphPad Prism 3.0 software.

Snap-frozen spinal cords were placed into 1 ml of TRIzol (Invitrogen) and homogenized in 2 ml of Tenbroeck glass grinders. RNA was isolated as previously described (43). DNA contamination was removed by treatment with DNase I for 30 min at 37°C (DNA-free kit; Ambion) and cDNA was synthesized from RNA using M-MLV Reverse Transcriptase (Invitrogen), oligo(dT) primers (Promega), and random primers (Promega). Quantitative real-time PCR was performed using 4 μl of cDNA and SYBR Green Master Mix (Applied Biosystems) in triplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems). PCR conditions were 10 min at 95°C followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Real-time primer sequences were as follows: GAPDH sense, 5′-CATGGCCTTCCGTGTTCCTA-3′ and antisense, 5′-ATGCCTGCTTCACCACCTTCT-3′ and JHMV nucleocapsid sense, 5′-CGCAGAGTATGGCGACGAT-3′ and antisense, 5′-GAGGTCCTAGTCTCGGCCTGTT-3′. Transcript levels were calculated relative to the housekeeping gene GAPDH using the following formula: 2[CT(GAPDH) − CT(target gene)] × 1000, where CT is determined as the threshold cycle at which the fluorescent signal becomes significantly higher than that of the background. Statistics were assessed using GraphPad Prism 3.0 software.

IFN-γ in spinal cord homogenate supernatants was measured by ELISA as previously described (50). Briefly, 96-well plates were coated overnight at 4°C with 100 μl of 1 μg/ml anti-IFN-γ (R4h6A2; BD Biosciences). Nonspecific binding was blocked with 10% FCS in PBS for 1.5 h before the addition of IFN-γ recombinant cytokine standard (XMG1.2; BD Biosciences) and samples. After a 2-h incubation at room temperature, bound IFN-γ was detected using biotinylated anti-IFN-γ (BD Biosciences) and avidin-peroxidase followed by 3,3′,5,5′-tetramethylbenzidine (TMB Reagent Set; BD Biosciences) 1 h later. Absorbance was read at 450 nm in a Bio-Rad model 680 microplate reader and analyzed using Microplate Manager 5.2 software (Bio-Rad).

Tissues were fixed in 10% formalin and embedded in paraffin. Spinal cords were examined for distribution of viral Ag and demyelination as previously described (35, 36, 37). Viral Ag was demonstrated by immunoperoxidase staining using anti-JHMV mAb J.3.3 specific for the C terminus of the viral nucleocaspid protein as the primary Ab and horse anti-mouse as secondary Ab (Vectastain ABC kit; Vector Laboratories). Demyelination was determined by staining with Luxol fast blue. Sections were scored in a blinded fashion and representative fields were identified based on the average score of all sections in each experimental group.

PD-1 expression on CD8 T cells is low or undetectable following clearance of peripheral infections, but remains high during chronic infection (51). JHMV infection is restricted to the CNS with no evidence for viral replication in lymphoid tissues (52). Furthermore infectious virus is reduced to undetectable levels by 2 wk postinfection (p.i.), but viral RNA persists at low levels (32, 36, 37, 38, 41, 50). PD-1 expression was assessed on CD8 T cells derived from the CNS, spleen, and CLN to compare regulation at the sites of priming and infection (32). CNS- infiltrating CD8 T cells expressed higher levels of PD-1 compared with CD8 T cells in the peripheral lymphoid organs (Fig. 1,A). Furthermore, virus-specific CD8 T cells expressed more PD-1 than tetramer-negative CD8 T cells (Fig. 1,B). Notably, the expression of PD-l on CNS-derived virus-specific CD8 T cells continued to increase following elimination of infectious virus from the CNS (Fig. 1,C) and remained elevated 6 wk p.i. (data not shown). By contrast, PD-1 expression remained relatively low on virus-specific CD8 T cells in peripheral lymphoid organs (Fig. 1 C), confirming the absence of ongoing peripheral Ag stimulation (52).

FIGURE 1.

CD8 T cells in the CNS express higher levels of PD-1 compared with peripheral CD8 T cells. Cells isolated from the spinal cord, CLN, and spleens of infected mice were analyzed for PD-1 expression by flow cytometry at the indicated days p.i. A, Representative histograms showing PD-1 on gated CD8 T cells from CLN (dashed line), spleen (gray line), and spinal cord (black solid line). B, Representative density plots depict staining with anti-PD-1 mAb (x-axis) and tetramer (y-axis) on spinal cord-infiltrating CD8 T cells. Numbers represent MFI of PD-1 staining (upper, tetramer-positive cells; lower, tetramer-negative cells). C, MFI of PD-1 expression on virus-specific CD8 T cells within spleen (□), CLN (), and spinal cord (▪). Data are representative of two independent experiments with three to four mice per time point.

FIGURE 1.

CD8 T cells in the CNS express higher levels of PD-1 compared with peripheral CD8 T cells. Cells isolated from the spinal cord, CLN, and spleens of infected mice were analyzed for PD-1 expression by flow cytometry at the indicated days p.i. A, Representative histograms showing PD-1 on gated CD8 T cells from CLN (dashed line), spleen (gray line), and spinal cord (black solid line). B, Representative density plots depict staining with anti-PD-1 mAb (x-axis) and tetramer (y-axis) on spinal cord-infiltrating CD8 T cells. Numbers represent MFI of PD-1 staining (upper, tetramer-positive cells; lower, tetramer-negative cells). C, MFI of PD-1 expression on virus-specific CD8 T cells within spleen (□), CLN (), and spinal cord (▪). Data are representative of two independent experiments with three to four mice per time point.

Close modal

Sustained expression of PD-1 on CNS-derived CD8 T cells (Fig. 1) and the loss of CD8 T cell effector function following initial virus control (40, 41) raised the possibility that PD-1:B7-H1 interactions may contribute to JHMV persistence. Oligodendroglia are primary targets of viral replication during acute infection and the reservoir of persisting virus (36). Therefore, expression of B7-H1 was compared on oligodendroglia and microglia from JHMV-infected proteolipid protein-GFP mice (Fig. 2,A). B7-H1 expression was undetectable by flow cytometry on naive oligodendroglia and microglia (Fig. 2,B). By day 5 p.i., 22% of oligodendroglia had up-regulated B7-H1. Expression levels further increased dramatically by day 7 p.i. and remained high through day 21 p.i. when infectious virus is undetectable in the CNS (32) and viral RNA has declined considerably (Table I). By contrast, few microglia expressed B7-H1 by day 5 p.i. and only transiently up-regulated B7-H1, reaching maximal levels at day 7 p.i. Expression subsequently diminished with only 7% expressing B7-H1 at day 21 p.i. (Fig. 2 B). These data show that peak percentages of B7-H1-positive cells were significantly greater in oligodendroglia compared with microglia during viral-induced encephalomyelitis.

FIGURE 2.

Oligodendroglia display enhanced and sustained B7-H1 expression relative to microglia. Cells from spinal cords of naive and infected animals were analyzed for GFP, CD45, and B7-H1 by flow cytometry at the indicated days p.i. A, Representative density plot showing CD45low microglia in region R1 and CD45GFP+ oligodendroglia in region R2 from the spinal cord of naive mice. B, Histograms depict B7-H1 expression (solid line) on oligodendroglia (left) and microglia (right). Isotype control staining is indicated by dashed lines. Data are representative of three independent experiments with four to five mice per time point and numbers within histograms represent mean percentages ± SEM of cells positive for B7-H1 expression relative to an isotype control.

FIGURE 2.

Oligodendroglia display enhanced and sustained B7-H1 expression relative to microglia. Cells from spinal cords of naive and infected animals were analyzed for GFP, CD45, and B7-H1 by flow cytometry at the indicated days p.i. A, Representative density plot showing CD45low microglia in region R1 and CD45GFP+ oligodendroglia in region R2 from the spinal cord of naive mice. B, Histograms depict B7-H1 expression (solid line) on oligodendroglia (left) and microglia (right). Isotype control staining is indicated by dashed lines. Data are representative of three independent experiments with four to five mice per time point and numbers within histograms represent mean percentages ± SEM of cells positive for B7-H1 expression relative to an isotype control.

Close modal
Table I.

Viral replication and IFN-γ within the CNS following JHMV infection

Days p.i.Viral NucleoproteinabIFN-γbc
1176 ± 300 0.6 ± 0.26 
7541 ± 2443 7.0 ± 0.98 
10 1674 ± 632 1.1 ± 0.11 
14 765 ± 246 0.9 ± 0.63 
21 49 ± 27 NDd 
Days p.i.Viral NucleoproteinabIFN-γbc
1176 ± 300 0.6 ± 0.26 
7541 ± 2443 7.0 ± 0.98 
10 1674 ± 632 1.1 ± 0.11 
14 765 ± 246 0.9 ± 0.63 
21 49 ± 27 NDd 
a

Relative spinal cord levels normalized to GAPDH mRNA.

b

Data presented as mean ± SEM.

c

IFN-γ (ng/spinal cord) assessed by ELISA.

d

ND, Not done.

Both PD-1 and B7-H1 can engage other related molecules (2), complicating the modulation of T cell activity. PD-L2, a second PD-1 ligand expressed on dendritic cells and macrophages, was not detected on either oligodendroglia or microglia from naive or infected mice by flow cytometry (data not shown). Activation induced up-regulation of constitutive B7-H1 expression on T cells (6, 7) also raised the possibility that B7-1, a member of the B7 family which also interacts with B7-H1 (53, 54) could contribute to inhibitory signaling. B7-H1 expression was indeed higher on CNS-infiltrating CD8 T cells compared with lymphoid organ CD8 T cells, confirming their activated phenotype (supplemental Fig. 16). Nevertheless, B7-1 expression was not induced on oligodendroglia during JHMV infection (supplemental Fig. 1). These data suggest that sustained expression of PD-l on virus-specific CD8 T cells, coincident with the sustained expression of B7-H1 on oligodendroglia, is likely to dampen CD8 T cell function.

The suppressive effect of B7-H1 is mediated through altered TCR signaling (2). Expression of surface MHC class I was thus monitored to assess the correlation of B7-H1 and MHC class I expression during the transition to viral persistence (Fig. 3). The percentage of MHC class I-positive oligodendroglia approximated that of B7-H1-positive cells at all time points except at day 7 p.i., suggesting an overall synchronous regulation of expression by oligodendroglia. Slight differences were evident by increased intensity of MHC class I expression throughout day 7 p.i., which was not evident for B7-H1 expression. By contrast, the frequency of MHC class I- expressing microglia exceeded B7-H1-expressing microglia by at least 3- to 4-fold and up to ∼9-fold by day 21 p.i. Overall, >60% of both oligodendroglia and microglia retained MHC class I expression during viral persistence (Fig. 3) despite a significant decline in IFN-γ levels between 7 and 21 days p.i. (Table I). Early MHC class I expression patterns during acute infection confirmed previous data (39); however, sustained MHC class I expression on oligodendroglia after clearance of infectious virus supports active immune suppression, rather than loss of Ag presentation, as a mechanism for viral persistence in oligodendroglia.

FIGURE 3.

Oligodendroglia and microglia sustain similar levels of MHC class I molecules. Histograms depict MHC class I expression (solid line) on oligodendroglia (left column) and microglia (right column) at the indicated days p.i. Isotype control staining is indicated by dashed lines. Data are representative of three independent experiments with four to five mice per time point. Numbers within histograms represent mean percentages ± SEM of cells positive for MHC class I expression relative to an isotype control.

FIGURE 3.

Oligodendroglia and microglia sustain similar levels of MHC class I molecules. Histograms depict MHC class I expression (solid line) on oligodendroglia (left column) and microglia (right column) at the indicated days p.i. Isotype control staining is indicated by dashed lines. Data are representative of three independent experiments with four to five mice per time point. Numbers within histograms represent mean percentages ± SEM of cells positive for MHC class I expression relative to an isotype control.

Close modal

B7-H1 expression can be induced by either type I or II IFN (6, 7, 8, 9, 10, 21). The delayed expression of MHC class I Ag-processing components relative to type I IFN (39) and concerted regulation of MHC class I and B7-H1 suggested that oligodendroglia may also require IFN-γ for B7-H1 expression. Oligodendroglia isolated from JHMV- infected IFN-γ−/− and IFNAR−/− mice were thus analyzed for B7-H1 expression. In the absence of IFN-γ, only 6% of oligodendroglia expressed B7-H1 at day 7 p.i, relative to 31% B7-H1-expressing oligodendroglia from infected wild-type (wt) mice (Fig. 4,A). By contrast, oligodendroglia isolated from IFNAR−/− infected mice at 7 days p.i. expressed comparable levels of B7-H1 relative to oligodendroglia isolated from wt mice (30% vs 28%, respectively; Fig. 4,B). B7-H1 expression on oligodendroglia from IFNAR−/−-infected animals could only be assessed until day 7 p.i. since the majority of JHMV-infected mice succumb by day 8 p.i (43). Nevertheless, B7-H1 up-regulation in the absence of type I IFN signaling is consistent with IFN-γ present in the CNS of infected IFNAR−/− mice (43). Contrasting with the control of B7-H1 expression, expression of PD-1 on CNS-derived CD8 T cells was IFN-γ independent (data not shown), confirming TCR- driven regulation of this inhibitory receptor (3). The apparent reliance of oligodendroglia on IFN-γ for expression of B7-H1 may be due to the relatively poor induction of type I IFN by coronaviruses (55, 56). Regulation of B7-H1 expression on oligodendroglia was thus examined following in vitro stimulation of oligodendroglia cells with IFN-β or IFN-γ. Untreated cells expressed little or no B7-H1 (Fig. 5), similar to oligodendroglia isolated from naive mice (Fig. 2,B). Both IFN-γ and IFN-β induced B7-H1 surface expression (Fig. 5); however, IFN-γ was superior to IFN-β in triggering B7-H1 expression as indicated by increased mean fluorescence intensity (MFI) (IFN-γ: MFI = 437; IFN-β: MFI = 164). This contrasts with MHC class I expression, which achieved higher levels after IFN-β compared with IFN-γ- mediated induction (IFN-γ: MFI = 147; IFN-β: MFI = 377; Fig. 5). PD-L2 expression was not detected, consistent with ex vivo- isolated oligodendroglia (data not shown). These data suggest that conditions in vivo dictate the timing and magnitude of B7-H1 expression in individual cell types, yet oligodendroglia have the capacity to express B7-H1 in response to both type I and type II IFN.

FIGURE 4.

B7-H1 expression on oligodendroglia is diminished in the absence of IFN-γ. Oligodendroglia from wt, IFN-γ−/−, and IFNAR−/−-infected and naive animals were assessed for B7-H1 expression by flow cytometry. Histograms in A illustrate the expression of B7-H1 on oligodendroglia from infected wt (gray solid line) and IFN-γ−/− animals (dashed line) at 7 p.i. B, Histograms depict the expression of B7-H1 on oligodendroglia from infected wt (gray solid line) and IFNAR−/− animals (dashed line) at 7 days p.i. Gray histograms in A and B represent expression of B7-H1 on naive oligodendroglia. Numbers within histograms represent mean percentages ± SEM of oligodendroglia positive for B7-H1 expression relative to naive animals (upper, IFN-γ−/− or IFNAR−/−; lower, wt). Data are representative of two or three independent experiments with three to four mice per time point.

FIGURE 4.

B7-H1 expression on oligodendroglia is diminished in the absence of IFN-γ. Oligodendroglia from wt, IFN-γ−/−, and IFNAR−/−-infected and naive animals were assessed for B7-H1 expression by flow cytometry. Histograms in A illustrate the expression of B7-H1 on oligodendroglia from infected wt (gray solid line) and IFN-γ−/− animals (dashed line) at 7 p.i. B, Histograms depict the expression of B7-H1 on oligodendroglia from infected wt (gray solid line) and IFNAR−/− animals (dashed line) at 7 days p.i. Gray histograms in A and B represent expression of B7-H1 on naive oligodendroglia. Numbers within histograms represent mean percentages ± SEM of oligodendroglia positive for B7-H1 expression relative to naive animals (upper, IFN-γ−/− or IFNAR−/−; lower, wt). Data are representative of two or three independent experiments with three to four mice per time point.

Close modal
FIGURE 5.

B7-H1 and MHC class I induction on oligodendroglia by IFN-γ and IFN-β. B7-H1 and MHC class I surface expression on N19 cells was analyzed by flow cytometry following treatment with either IFN-γ (5 ng/ml) or IFN-β (3 ng/ml) for 24 h. Histograms depict the expression of B7-H1 or MHC class I as indicated. B7-H1 expression after IFN treatment is indicated by a black histogram, while MHC class I expression is indicated by a gray histogram. B7-H1 or MHC class I expression on untreated N19 cells is indicated by a solid black line and isotype control staining by the dashed line. Numbers in the right-hand corner indicate the MFI of B7-H1 or MHC class I on IFN-treated cells. Data are representative of three independent experiments.

FIGURE 5.

B7-H1 and MHC class I induction on oligodendroglia by IFN-γ and IFN-β. B7-H1 and MHC class I surface expression on N19 cells was analyzed by flow cytometry following treatment with either IFN-γ (5 ng/ml) or IFN-β (3 ng/ml) for 24 h. Histograms depict the expression of B7-H1 or MHC class I as indicated. B7-H1 expression after IFN treatment is indicated by a black histogram, while MHC class I expression is indicated by a gray histogram. B7-H1 or MHC class I expression on untreated N19 cells is indicated by a solid black line and isotype control staining by the dashed line. Numbers in the right-hand corner indicate the MFI of B7-H1 or MHC class I on IFN-treated cells. Data are representative of three independent experiments.

Close modal

PD-1:B7-H1 interactions negatively regulate T cell activation, resulting in suppressed cytokine secretion (8, 9, 15, 20, 21, 22). To determine whether B7-H1 expression on oligodendroglia can dampen CD8 T cell function, IFN-γ- treated oligodendroglia were tested as CD8 T cell stimulators in the presence or absence of anti-B7-H1 mAb. Blockade of B7-H1 significantly enhanced CD8 T cell IFN-γ secretion by virus-specific memory CD8 T cells (Fig. 6). These data demonstrate that PD-1:B7-H1 interactions between oligodendroglia and CD8 T cells down-regulates IFN-γ secretion and support their role in dampening T cell effector functions in vivo.

FIGURE 6.

B7-H1 expression on oligodendroglia alters IFN-γ secretion by CD8 T cells. IFN-γ-treated, peptide-coated N19 cells were cocultured with memory T cells from JHMV-immunized mice for 96 h. Anti-B7-H1 Ab was added at a final concentration of 10 μg/ml at the start of culture. Culture supernatants were collected and assessed for IFN-γ secretion by ELISA. Data are representative of two independent experiments. Statistically significant differences between the two experimental groups, determined by unpaired t test, are denoted by ∗∗∗, p < 0.001.

FIGURE 6.

B7-H1 expression on oligodendroglia alters IFN-γ secretion by CD8 T cells. IFN-γ-treated, peptide-coated N19 cells were cocultured with memory T cells from JHMV-immunized mice for 96 h. Anti-B7-H1 Ab was added at a final concentration of 10 μg/ml at the start of culture. Culture supernatants were collected and assessed for IFN-γ secretion by ELISA. Data are representative of two independent experiments. Statistically significant differences between the two experimental groups, determined by unpaired t test, are denoted by ∗∗∗, p < 0.001.

Close modal

Sustained expression of B7-H1 by oligodendroglia supports a role for PD-1:B7-H1 interactions in hindering JHMV clearance from the CNS. To verify this notion, JHMV replication was compared in wt and B7-H1−/− mice. The onset of clinical symptoms was comparable in wt and B7-H1−/− mice (Fig. 7,A); however, severity was substantially increased in infected B7-H1−/− mice after day 10 p.i. Similar levels of viral mRNA in the CNS of infected wt and B7-H1−/− mice at day 7 p.i. indicated that B7-H1 exerted no impact on initial virus replication (Fig. 7,B). Consistent with a negative influence of B7-H1 expression on virus clearance, viral mRNA was significantly reduced in B7-H1−/− mice by day 10 p.i. This was also supported by reduced numbers of infected cells within the CNS in infected B7-H1−/− mice vs wt mice (Fig. 8). Although the number of infected cells was reduced in the CNS of B7-H1−/− mice, no differences in viral tropism were noted comparing wt and B7-H1−/− mice. Oligodendroglia represented the predominant cell expressing viral Ag. Furthermore, enhanced virus clearance from the CNS of B7-H1−/− mice did not alter the severity of demyelination (Fig. 8). These data suggest that enhanced morbidity was not directly correlated with oligodendroglia damage imposed by enhanced CD8 T cell effector function.

FIGURE 7.

Increased disease severity associated with rapid virus control in B7-H1−/− mice. A, Infected wt and B7-H1−/− mice (n = 10/group) were scored for clinical symptoms as described in Materials and Methods. Clinical scores at the indicated time points are cumulative and include morbidity. Data are expressed as the mean ± SEM. B, Levels of viral mRNA in spinal cords determined by real-time PCR. Data are expressed as the mean ± SEM transcript level of viral mRNA relative to GAPDH mRNA. Statistically significant differences between infected wt and B7-H1−/− mice, determined by unpaired t test, are denoted by ∗, p < 0.05; ∗∗, p < 0.005; and ∗∗∗, p < 0.001.

FIGURE 7.

Increased disease severity associated with rapid virus control in B7-H1−/− mice. A, Infected wt and B7-H1−/− mice (n = 10/group) were scored for clinical symptoms as described in Materials and Methods. Clinical scores at the indicated time points are cumulative and include morbidity. Data are expressed as the mean ± SEM. B, Levels of viral mRNA in spinal cords determined by real-time PCR. Data are expressed as the mean ± SEM transcript level of viral mRNA relative to GAPDH mRNA. Statistically significant differences between infected wt and B7-H1−/− mice, determined by unpaired t test, are denoted by ∗, p < 0.05; ∗∗, p < 0.005; and ∗∗∗, p < 0.001.

Close modal
FIGURE 8.

Viral Ag in the CNS of infected wt and B7-H1−/− mice. Upper panels, Virus-infected cells in spinal cord white matter tracks of infected wt and B7-H1−/− mice at 10 days p.i. Arrows indicate Ag-positive cells with morphology consistent with oligodendroglia. Lower panels, Demyelination in spinal cords of infected wt and B7-H1−/− mice at day 10 p.i. ∗, Areas of demyelination within the white matter, while ∗∗, normal adjacent white matter. Scale bar, 100 μm.

FIGURE 8.

Viral Ag in the CNS of infected wt and B7-H1−/− mice. Upper panels, Virus-infected cells in spinal cord white matter tracks of infected wt and B7-H1−/− mice at 10 days p.i. Arrows indicate Ag-positive cells with morphology consistent with oligodendroglia. Lower panels, Demyelination in spinal cords of infected wt and B7-H1−/− mice at day 10 p.i. ∗, Areas of demyelination within the white matter, while ∗∗, normal adjacent white matter. Scale bar, 100 μm.

Close modal

Restrained T cell activity attributed to PD-1:B7-H1 interactions has been proposed to contribute to numerous persistent infections (19, 20, 25, 26, 27, 51). However, expression of modulatory molecules on susceptible cells during the course of infection, especially within specialized tissues such as the CNS, is not well characterized. The present study examined the role of PD-1:B7-H1 interactions in contributing to immune evasion of a glialtropic coronavirus in the CNS. CD8 T cells are critical in restraining acute viral replication, yet viral RNA persists primarily in oligodendroglia, despite CD8 T cell retention within the CNS (40, 41, 57). CNS-infiltrating virus-specific CD8 T cells expressed PD-1, which increased over time, even after infectious virus had been cleared from the CNS. Importantly, B7-H1 was up-regulated on both virus-susceptible microglia and oligodendroglia, yet the expression patterns differed. Although B7-H1 was only transiently expressed at moderate levels on microglia, it was abundantly expressed by the vast majority of oligodendroglia. Expression of B7-H1 on astrocytes was similar to microglia with maximal expression on 39% of the cells at day 7 p.i., which diminished considerably by day 10 p.i. (data not shown). Elevated B7-H1 expression between days 7 and 10 p.i. correlated with lymphocyte accumulation within the CNS. However, the increased magnitude and sustained B7-H1 expression by oligodendroglia support a role for PD-1:B7-H1 in dampening CD8 T cell function early as well as maintaining inhibitory functions specifically toward oligodendroglia, thus favoring persistence in this cell type. Notably, only a minority of oligodendroglia are infected in wt mice (36), suggesting that the majority of B7-H1-positive oligodendroglia are uninfected.

Type I and II IFNs up-regulate B7-H1 on various cell types (6, 7, 8, 9, 10, 21). However, the relative contribution of each IFN may be cell type specific. A dominant contribution of IFN-γ to B7-H1 expression by oligodendroglia in vivo was supported by considerably decreased expression in infected IFN-γ-deficient mice. By contrast, the frequency of B7-H1 expressing microglia decreased from 19% in wt to 9% in IFN-γ-deficient mice (data not shown). A direct affect of type I IFN on B7-H1 expression by glia in vivo could not be assessed due to the presence of IFN-γ in the CNS of infected IFNAR−/− mice (43). Indeed, B7-H1 expression on oligodendroglia from IFNAR−/−-infected mice was comparable to oligodendroglia derived from wt mice, suggesting a minor, if any contribution, of type I IFN to B7-H1 expression on oligodendroglia in vivo. These observations contrast with the prominent role of IFN-β in up-regulating B7-H1 on neurons during rabies virus infection (10). The poor type I IFN-inducing phenotype of coronaviruses may contribute to the dominant effects of IFN-γ in the JHMV model (32, 55, 56); however, exogenous IFN-γ was also superior to IFN-β in inducing B7-H1 expression on oligodendroglia.

The balance between MHC class I recognition and B7-H1 engagement is no doubt critical in determining the magnitude of antiviral functions, as the inhibitory effect of PD-1:B7-H1 acts through MHC class I-mediated TCR signaling. Similar to B7-H1, MHC class I up-regulation on oligodendroglia is also dependent on IFN-γ and is delayed relative to microglia following JHMV infection (39). Sparse B7-H1 expression relative to MHC class I expression on microglia may only modestly affect CD8 T cell function, while its prominent expression by oligodendroglia is likely to impose more restrictions on CD8 T cell function. The present report demonstrates that both B7-H1 and MHC class I expression are sustained at high levels on oligodendroglia following infection, while only MHC class I is maintained on microglia. The rapid drop in IFN-γ within the CNS after 7 days p.i. suggests that persistent B7-H1 expression on oligodendroglia may be sustained by limited focal IFN-γ production. These observations suggest a model in which productive engagement of CD8 T cells by infected microglia triggers IFN-γ production, leading not only to enhanced MHC class I expression, but also to B7-H1 up-regulation in select cell types. In addition to its antiviral function, IFN-γ may thus simultaneously promote persistence. Although the synchronous expression of MHC class I and B7-H1 on oligodendroglia appears paradoxical, it may provide a mechanism to sequester CD8 T cells to sites of chronic Ag presentation, while limiting immune pathology.

Several other members of the B7 family which inhibit T cell function have been excluded from contributing to oligodendrocyte-mediated inhibition of CD8 T cell function. PD-L2 expression remained undetectable on microglia and oligodendroglia from JHMV-infected animals or after IFN-γ or IFN-β stimulation of oligodendroglia in vitro (data not shown), consistent with its restricted expression by dendritic cells and macrophages (7). Also, oligodendroglia did not express B7-1, which has recently been found to deliver inhibitory signals following interactions with B7-H1 (53, 54) similar to PD-1:B7-H1 interactions. CD8 T cells from the JHMV-infected CNS did however express B7-H1, consistent with elevated B7-H1 expression on activated T cells (6, 7). Although the absence of B7-1 expression on oligodendroglia negates B7-H1-mediated effects on CD8 T cells by this cell type, interactions with B7-1-expressing microglia cannot be excluded. Oligodendroglia B7-H1-mediated engagement of B7-1 on T cells (58, 59) also remains possible.

Studies with various non-neuronal cell lines document that blockade of PD-1:B7-H1 signaling enhanced IFN-γ secretion by T cells (8, 9, 15, 20, 21, 22). Similarly, blockade of B7-H1 significantly increased IFN-γ secretion by JHMV-specific CD8 T cells, supporting sustained B7-H1 expression by oligodendroglia as a mechanism contributing to dampened CD8 T responses in the CNS (40, 41, 57). The inhibitory actions of B7-H1 were indeed supported by significantly lower levels of viral mRNA and Ag in the CNS of B7-H1−/− mice compared with wt mice at 10 days p.i. Comparable viral mRNA levels at day 7 p.i. in both mouse groups combined with the absence of class II expression on oligodendrocytes (60) strongly implicates enhanced CD8 T cell effector function in mediating the more effective virus control. Treatment with anti-PD-1- or anti-B7-H1- blocking Ab can restore CD8 T cell effector function in models of chronic infection (6, 27, 40). However, the use of therapeutic blocking Ab approaches to inhibit PD-1:B7-H1 interactions during CNS infections may prove challenging due to obstacles imposed by the blood-brain barrier. Importantly, PD-1 and its ligand also regulate immunopathology (13, 22, 49, 61, 62). For example, more rapid clearance of adenovirus from PD-1-deficient mice is counteracted by increased hepatocellular injury (22). Similarly, the absence of PD-1 or B7-H1 in experimental autoimmune encephalomyelitis results in increased disease severity (61). During JHMV infection, disease severity was also exacerbated in the absence of B7-H1 despite lower virus load, supporting a protective role of B7-H1 expression in CNS tissues. The observation that enhanced morbidity did not correlate with exacerbated demyelination warrants future investigation into the pathogenic mechanisms. Nevertheless, these data argue that PD-1:B7-H1 interactions limit excessive immune-mediated damage at the cost of viral persistence. By contrast, B7-H1 deficiency rescues mice from fatal rabies virus infection (10). Although this phenotype was associated with early prevention of viral spread to the CNS, the mechanisms were not investigated.

In summary, expression of B7-H1 by oligodendroglia, in addition to its expression by astrocytes (11, 13), microglia (12, 13), and neurons (10) provides accumulating evidence for B7-H1 as a critical immune modulator in CNS disease. The distinct regulation of MHC class I and B7-H1 by microglia and oligodendroglia emphasize the unique propensity of CNS resident cells to interact with CD8 T cells during the course of infection. Specifically, the early expression of B7-H1 by oligodendroglia may balance CD8 T cell-mediated antiviral mechanisms with preservation of myelin and neuronal function.

We sincerely thank Dr. Hideo Yagita from Juntendo University School of Medicine (Tokyo, Japan) for the MIH6 anti-B7-H1 Ab.

The authors have no financial conflict of interest.

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 National Institutes of Health Grants NS18146 and AI47249.

5

Abbreviations used in this paper: PD-1, programmed death-1; CLN, cervical lymph node; JHMV, neurotropic JHM strain of mouse hepatitis virus; MFI, mean fluorescence intensity; p.i., postinfection; wt, wild type; CT, threshold cycle.

6

The online version of this article supplemental material.

1
Gloor, S. M., M. Wachtel, M. F. Bolliger, H. Ishihara, R. Landmann, K. Frei.
2001
. Molecular and cellular permeability control at the blood-brain barrier.
Brain Res. Rev.
36
:
258
-264.
2
Keir, M. E., M. J. Butte, G. J. Freeman, A. H. Sharpe.
2008
. PD-1 and its ligands in tolerance and immunity.
Annu. Rev. Immunol.
26
:
677
-704.
3
Agata, Y., A. Kawasaki, H. Nishimura, Y. Ishida, T. Tsubata, H. Yagita, T. Honjo.
1996
. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes.
Int. Immunol.
8
:
765
-772.
4
Petrovas, C., J. P. Casazza, J. M. Brenchley, D. A. Price, E. Gostick, W. C. Adams, M. L. Precopio, T. Schacker, M. Roederer, D. C. Douek, R. A. Koup.
2006
. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection.
J. Exp. Med.
203
:
2281
-2292.
5
Golden-Mason, L., J. Klarquist, A. S. Wahed, H. R. Rosen.
2008
. Cutting edge: programmed death-1 expression is increased on immunocytes in chronic hepatitis C virus and predicts failure of response to antiviral therapy: race-dependent differences.
J. Immunol.
180
:
3637
-3641.
6
Maier, H., M. Isogawa, G. J. Freeman, F. V. Chisari.
2007
. PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver.
J. Immunol.
178
:
2714
-2720.
7
Yamazaki, T., H. Akiba, H. Iwai, H. Matsuda, M. Aoki, Y. Tanno, T. Shin, H. Tsuchiya, D. M. Pardoll, K. Okumura, et al
2002
. Expression of programmed death 1 ligands by murine T cells and APC.
J. Immunol.
169
:
5538
-5545.
8
Rodig, N., T. Ryan, J. A. Allen, H. Pang, N. Grabie, T. Chernova, E. A. Greenfield, S. C. Liang, A. H. Sharpe, A. H. Lichtman, G. J. Freeman.
2003
. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis.
Eur. J. Immunol.
33
:
3117
-3126.
9
Usui, Y., Y. Okunuki, T. Hattori, T. Kezuka, H. Keino, N. Ebihara, S. Sugita, M. Usui, H. Goto, M. Takeuchi.
2008
. Functional expression of B7H1 on retinal pigment epithelial cells.
Exp. Eye Res.
86
:
52
-59.
10
Lafon, M., F. Mergret, S. Meuth, O. Simon, M. L. Velandia-Romero, M. Lafage, L. Chen, L. Alexopoulou, R. A. Flavell, C. Prehaud, H. Wiendl.
2008
. Detrimental contribution of the immuno-inhibitor B7-H1 to rabies virus encephalitis.
J. Immunol.
180
:
7506
-7515.
11
Lipp, M., C. Brandt, F. Dehghani, E. Kwidzinski, I. Bechmann.
2007
. PD-L1 (B7-H1) regulation in zones of axonal degeneration.
Neurosci. Lett.
425
:
156
-161.
12
Magnus, T., B. Schreiner, T. Korn, C. Jack, H. Guo, J. Antel, I. Ifergan, L. Chen, F. Bischof, A. Bar-Or, H. Wiendl.
2005
. Microglia expression of the B7 family member B7 homolog 1 confers strong immune inhibition: implications for immune responses and autoimmunity in the CNS.
J. Neurosci.
25
:
2537
-2546.
13
Salama, A. D., T. Chitnis, J. Imitola, M. J. Ansari, H. Akiba, F. Tushima, M. Azuma, H. Yagita, M. H. Sayegh, S. J. Khoury.
2003
. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis.
J. Exp. Med.
198
:
71
-78.
14
Benedict, C. A., A. Loewendorf, Z. Garcia, B. R. Blazar, E. M. Janssen.
2008
. Dendritic cell programming by cytomegalovirus stunts naive T cell responses via the PD-L1/PD-1 pathway.
J. Immunol.
180
:
4836
-4847.
15
Jeong, H. Y., Y. J. Lee, S. K. Seo, S. W. Lee, S. J. Park, J. N. Lee, H. S. Sohn, S. Yao, L. Chen, I. Choi.
2008
. Blocking of monocyte-associated B7-H1 (CD274) enhances HCV-specific T cell immunity in chronic hepatitis C infection.
J. Leukocyte Biol.
83
:
755
-764.
16
Sester, U., D. Presser, J. Dirks, B. C. Gärtner, H. Köhler, M. Sester.
2008
. PD-1 expression and IL-2 loss of cytomegalovirus- specific T cells correlates with viremia and reversible functional anergy.
Am. J. Transplant.
8
:
1486
-1497.
17
Wherry, E. J., S. J. Ha, S. M. Kaech, W. N. Haining, S. Sarkar, V. Kalia, S. Subramaniam, J. N. Blattman, D. L. Barber, R. Ahmed.
2007
. Molecular signature of CD8+ T cell exhaustion during chronic viral infect.
Immunity
27
:
670
-684.
18
Wong, R. M., R. R. Scotland, R. L. Lau, C. Wang, A. J. Korman, W. M. Kast, J. S. Weber.
2007
. Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs.
Int. Immunol.
19
:
1223
-1234.
19
Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy, E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, et al
2006
. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression.
Nature
443
:
350
-354.
20
Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette, M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, et al
2006
. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction.
Nat. Med.
12
:
1198
-1202.
21
Schreiner, B., M. Mitsdoerffer, B. C. Kieseier, L. Chen, H. P. Hartung, M. Weller, H. Wiendl.
2004
. Interferon-β enhances monocyte and dendritic cell expression of B7–H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis.
J. Neuroimmunol.
155
:
172
-182.
22
Iwai, Y., S. Terawaki, M. Ikegawa, T. Okazaki, T. Honjo.
2003
. PD-1 inhibits antiviral immunity at the effector phase in the liver.
J. Exp. Med.
198
:
39
-50.
23
Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, R. Ahmed.
2003
. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distant stages of functional impairment.
J. Virol.
77
:
4911
-4927.
24
Zajac, Y. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, R. Ahmed.
1998
. Viral immune evasion due to persistence of activated T cells without effector function.
J. Exp. Med.
188
:
2205
-2213.
25
Peng, G., S. Li, W. Wu, X. Tan, Y. Chen, Z. Chen.
2008
. PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients.
Mol. Immunol.
45
:
963
-970.
26
Penna, A., M. Pilli, A. Zerbini, A. Orlandini, S. Mezzadri, L. Sacchelli, G. Missale, C. Ferrari.
2007
. Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection.
Hepatology
45
:
588
-601.
27
Radziewicz, H., C. C. Ibegbu, M. L. Fernandez, K. A. Workowski, K. Obideen, M. Wehbi, H. L. Hanson, J. P. Steinberg, D. Masopust, E. J. Wherry, et al
2007
. Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.
J. Virol.
81
:
2545
-2553.
28
Onlamoon, N., K. Rogers, A. E. Mayne, K. Pattanapanyasat, K. Mori, F. Villinger, A. A. Ansari.
2008
. Soluble PD-1 rescues the proliferative response of simian immunodeficiency virus-specific CD4 and CD8 T cells during chronic infection.
Immunology
124
:
277
-293.
29
Wintterle, S., B. Schreiner, M. Mitsdoerffer, D. Schneider, L. Chen, R. Meyermann, M. Weller, H. Wiendl.
2003
. Expression of the B7-related molecule B7–H1 by glioma cells: a potential mechanism of immune paralysis.
Cancer Res.
63
:
7462
-7467.
30
Dong, H., S. E. Strome, D. R. Salomao, H. Tamura, F. Hirano, D. B. Flies, P. C. Roche, J. Lu, G. Zhu, K. Tamada, et al
2002
. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.
Nat. Med.
8
:
793
-800.
31
Suvas, S., A. K. Azkur, B. T. Rouse.
2006
. Qa-1b and CD94-NKG2a interaction regulate cytolytic activity of herpes simplex virus-specific memory CD8+ T cells in the latently infected trigeminal ganglia.
J. Immunol.
176
:
1703
-1711.
32
Bergmann, C. C., T. E. Lane, S. A. Stohlman.
2006
. Coronavirus infection of the central nervous system: host-virus stand-off.
Nat. Rev. Microbiol.
4
:
121
-132.
33
Marten, N. W., S. A. Stohlman, C. C. Bergmann.
2001
. MHV infection of the CNS: mechanism of immune-mediated control.
Viral Immunol.
14
:
1
-18.
34
Wang, F. I., S. A. Stohlman, J. O. Fleming.
1990
. Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated.
J. Neuroimmunol.
30
:
31
-41.
35
Bergmann, C. C., B. Parra, D. R. Hinton, C. Ramakrishna, K. C. Dowdell, S. A. Stohlman.
2004
. Perforin and γ interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells.
J. Virol.
78
:
1739
-1750.
36
González, J. M., C. C. Bergmann, C. Ramakrishna, D. R. Hinton, R. Atkinson, J. Hoskin, W. B. Macklin, S. A. Stohlman.
2006
. Inhibition of interferon-γ signaling in oligodendroglia delays coronavirus clearance without altering demyelination.
Am. J. Pathol.
168
:
796
-804.
37
Parra, B., D. R. Hinton, N. W. Marten, C. C. Bergmann, M. T. Lin, C. S. Yang, S. A. Stohlman.
1999
. IFN-γ is required for viral clearance from central nervous system oligodendroglia.
J. Immunol.
162
:
1641
-1647.
38
Lin, M. T., S. A. Stohlman, D. R. Hinton.
1997
. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis.
J. Virol.
71
:
383
-391.
39
Malone, K. E., S. A. Stohlman, C. Ramakrishna, W. B. Macklin, C. C. Bergmann.
2008
. Induction of class I antigen processing components in oligodendroglia and microglia during viral encephalomyelitis.
Glia
56
:
426
-435.
40
Ramakrishna, C., R. A. Atkinson, S. A. Stohlman, C. C. Bergmann.
2006
. Vaccine-induced memory CD8+ T cells cannot prevent central nervous system virus reactivation.
J. Immunol.
176
:
3062
-3069.
41
Bergmann, C. C., J. D. Altman, D. Hinton, S. A. Stohlman.
1999
. Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.
J. Immunol.
163
:
3379
-3387.
42
Fuss, B., B. Mallon, T. Phan, C. Ohlemeyer, F. Kirchhoff, A. Nishiyama, W. B. Macklin.
2000
. Purification and analysis of in vivo-differentiated oligodendrocytes expressing the green fluorescent protein.
Dev. Biol.
218
:
259
-274.
43
Ireland, D. D., S. A. Stohlman, D. R. Hinton, R. Atkinson, C. C. Bergmann.
2008
. Type I interferons are essential in controlling neurotropic coronavirus infection irrespective of functional CD8 T cells.
J. Virol.
82
:
300
-310.
44
Dong, H., G. Zhu, K. Tamada, D. B. Flies, J. M. van Deursen, L. Chen.
2004
. B7-H1 determines accumulation and deletion of intrahepatic CD8+ T lymphocytes.
Immunity
20
:
327
-336.
45
Hamo, L., S. A. Stohlman, M. Otto-Duessel, C. C. Bergmann.
2007
. Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis.
Glia
55
:
1169
-1177.
46
Fleming, J. O., M. D. Trousdale, F. A. el-Zaatari, S. A. Stohlman, L. P. Weiner.
1996
. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.
J. Virol.
58
:
869
-875.
47
Schachner, M., S. K. Kim, R. Zehnle.
1981
. Developmental expression in central and peripheral nervous system of oligodendrocyte cell surface antigens (O antigens) recognized by monoclonal antibodies.
Dev. Biol.
83
:
328
-338.
48
Foster, L. M., T. Phan, A. N. Verity, D. Bredesen, A. T. Campagnoni.
1993
. Generation and analysis of normal and shiverer temperature-sensitive immortalized cell lines exhibiting phenotypic characteristics of oligodendrocytes at several stages of differentiation.
Dev. Neurosci.
15
:
100
-109.
49
Kanai, T., T. Totsuka, K. Uraushihara, S. Makita, T. Nakamura, K. Koganei, T. Fukushima, H. Akiba, H. Yagita, K. Okumura, et al
2003
. Blockade of B7-H1 suppresses the development of chronic intestinal inflammation.
J. Immunol.
171
:
4156
-4163.
50
Zuo, J., S. A. Stohlman, J. B. Hoskin, D. R. Hinton, R. Atkinson, C. C. Bergmann.
2006
. Mouse hepatitis virus pathogenesis in the central nervous system is independent of IL-15 and natural killer cells.
Virology
350
:
206
-215.
51
Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe, J G. Freeman, R. Ahmed.
2006
. Restoring function in exhausted CD8 T cells during chronic viral infection.
Nature
439
:
682
-687.
52
Marten, N. W., S. A. Stohlman, J. Zhou, C. C. Bergmann.
2003
. Kinetics of virus-specific CD8+ -T-cell expansion and trafficking following central nervous system infection.
J. Virol.
77
:
2775
-2778.
53
Butte, M. J., V. Pena-Cruz, M. J. Kim, G. J. Freeman, A. H. Sharpe.
2008
. Interaction of human PD-L1 and B7-1.
Mol. Immunol.
45
:
3567
-3572.
54
Butte, M. J., M. E. Keir, T. B. Phamduy, A. H. Sharpe, G. J. Freeman.
2007
. Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses.
Immunity
27
:
111
-122.
55
Roth-Cross, J. K., S. J. Bender, S. R Weiss.
2008
. Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia.
J. Virol.
82
:
9829
-9838.
56
Zhou, H., S. Perlman.
2007
. Mouse hepatitis virus does not induce β interferon synthesis and does not inhibit its induction by double-stranded RNA.
J. Virol.
81
:
568
-574.
57
Ramakrishna, C., S. A. Stohlman, R. D. Atkinson, M. J. Shlomchik, C. C. Bergmann.
2002
. Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells.
J. Immunol.
168
:
1204
-1211.
58
Taylor, P. A., C. J. Lees, S. Fournier, J. P. Allison, A. H. Sharpe, B. R. Blazar.
2004
. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions.
J. Immunol.
172
:
34
-39.
59
Sun, Z. W., Y. H. Qiu, Y. J. Shi, R. Tao, J. Chen, Y. Ge, Y. M. Hu, H. B. Ma, Q. Shi, X. G. Zhang.
2005
. Time courses of B7 family molecules expressed on activated T-cells and their biological significance.
Cell. Immunol.
236
:
146
-153.
60
Redwine, J. M., M. J. Buchmeier, C. F. Evans.
2001
. In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection.
Am. J. Pathol.
159
:
1219
-1224.
61
Carter, L. L., M. W. Leach, M. L. Azoitei, J. Cui, J. W. Pelker, J. Jussif, S. Benoit, G. Ireland, D. Luxenberg, G. R. Askew, et al
2007
. PD-1/PD-L1, but not PD-1/PD-L2, interactions regulate the severity of experimental autoimmune encephalomyelitis.
J. Neuroimmunol.
182
:
124
-134.
62
Jun, H., S. K. Seo, H. Y. Jeong, H. M. Seo, G. Zhu, L. Chen, I. H. Choi.
2005
. B7-H1 (CD274) inhibits the development of herpetic stromal keratitis (HSK).
FEBS Lett.
579
:
6259
-6264.