Enhanced CD8 T-cell anti-viral function and clinical disease in B7-H1-deficient mice requires CD4 T cells during encephalomyelitis
© Phares et al.; licensee BioMed Central Ltd. 2012
Received: 31 July 2012
Accepted: 3 December 2012
Published: 14 December 2012
Anti-viral CD8 T-cell activity is enhanced and prolonged by CD4 T-cell-mediated help, but negatively regulated by inhibitory B7-H1 interactions. During viral encephalomyelitis, the absence of CD4 T cells decreases CD8 T cell activity and impedes viral control in the central nervous system (CNS). By contrast, the absence of B7-H1 enhances CD8 T-cell function and accelerates viral control, but increases morbidity. However, the relative contribution of CD4 T cells to CD8 function in the CNS, in the absence of B7-H1, remains unclear.
Wild-type (WT) and B7-H1−/− mice were infected with a gliatropic coronavirus and CD4 T cells depleted to specifically block T helper function in the CNS. Flow cytometry and gene expression analysis of purified T-cell populations from lymph nodes and the CNS was used to directly monitor ex vivo T-cell effector function. The biological affects of altered T-cell responses were evaluated by analysis of viral control and spinal-cord pathology.
Increased anti-viral activity by CD8 T cells in the CNS of B7-H1−/− mice was lost upon depletion of CD4 T cells; however, despite concomitant loss of viral control, the clinical disease was less severe. CD4 depletion in B7-H1−/− mice also decreased inducible nitric oxide synthase expression by microglia and macrophages, consistent with decreased microglia/macrophage activation and reduced interferon (IFN)-γ. Enhanced production of IFN-γ, interleukin (IL)-10 and IL-21 mRNA was seen in CD4 T cells from infected B7-H1−/− compared with WT mice, suggesting that over-activated CD4 T cells primarily contribute to the increased pathology.
The local requirement of CD4 T-cell help for CD8 T-cell function is not overcome if B7-H1 inhibitory signals are lost. Moreover, the increased effector activity by CD8 T cells in the CNS of B7-H1−/− mice is attributable not only to the absence of B7-H1 upregulation on major histocompatibility complex class I-presenting resident target cells, but also to enhanced local CD4 T-cell function. B7-H1-mediated restraint of CD4 T-cell activity is thus crucial to dampen both CD8 T-cell function and microglia/macrophage activation, thereby providing protection from T-cell-mediated bystander damage.
KeywordsCentral nervous system Encephalomyelitis CD4+ and CD8+ T cells Gliatropic coronavirus Inflammation Axonal damage
The magnitude, quality and longevity of CD8 T-cell effector function is positively regulated by CD4 T cells, and negatively regulated by various T-cell inhibitory molecules. CD4 T cells augment CD8 T-cell activation and expansion, directly through the production of cytokines or indirectly by licensing dendritic cells (DCs) in draining lymph nodes [1, 2]. Moreover, CD4 T cells can further enhance the primary anti-viral responses of CD8 T cells and promote their survival in the target organ [3–8]. This function is especially crucial in sustaining CD8 T-cell activity during prolonged and chronic infections. Paradoxically however, both CD4 and CD8 T cells upregulate numerous inhibitory molecules upon extended exposure to antigen to counterbalance over-exuberant, and potentially damaging, T-cell activity. Negative regulation by T-cell engagement of inhibitory ligands allows customized fine-tuning of T-cell function and mobility by the respective antigen-presenting cells (APCs) in the local environment. Among the components regulating the delicate balance between protective and detrimental immunity is programmed death (PD)-1, which dampens T-cell proliferation, cytokine production, and cytolytic activity following interaction with its ligand B7-H1.
The ongoing regulation of T cells and their adaptation to the local environment is most apparent during persistent infections, when CD4 T cells are essential to prolong CD8 T-cell function and survival [9–11]; however interactions between inhibitory receptors and their ligands dampen anti-viral function [11–15]. This paradigm also applies to encephalomyelitis induced by the sub-lethal gliatropic JHM strain of mouse hepatitis virus (JHMV). In this model, T cells control acute virus replication using both perforin-mediated and interferon (IFN)-γ-mediated mechanisms [16–19]; however, CD8 T-cell function rapidly wanes, allowing persistent infection . Furthermore, T-cell activity is associated with immune-mediated demyelination, which is sustained throughout the viral persistence . CD8 T cells are primary adaptive anti-viral effectors, but CD4 T cells play a vital supportive role, and may also directly contribute to viral control [8, 16–19]. Depletion of CD4 T cells at distinct times relative to infection showed that CD4 T cells not only enhance peripheral CD8 T-cell priming/expansion, but further promote CD8 T-cell function locally within the central nervous system (CNS) . CD8 T cells deprived of CD4 T-cell help within the CNS (designated ‘unhelped’ CD8 T cells), have diminished effector function and are unable to control virus replication . Recent analysis of the contribution of inhibitory interactions during JHMV pathogenesis further showed that virus-specific CD8 T cells in the CNS express high levels of PD-1 . Moreover, oligodendroglia, which are prominent targets of infection, strongly upregulate the ligand B7-H1 in response to IFN-γ during infection . In a previous study, the dampening effects of PD-1:B7-H1 interaction were evident by increased IFN-γ and granzyme B production by CNS-infiltrating CD8 T cells, coincident with accelerated virus control and decreased viral persistence in JHMV-infected B7-H1 deficient (B7-H1−/−) mice . Conversely, however, the extent of axonal damage was exacerbated, suggesting that B7-H1 mediates protection from immune pathology and mortality . Oligodendrocytes were shown to upregulate major histocompatibility complex (MHC) class I, but not MHC class II during JHMV infection , thus the improved viral control was attributed to enhanced CD8 T-cell activity. However, demyelination was similar to infected wild-type (WT) mice , suggesting that the absence of B7-H1 did not overtly increase the vulnerability of oligodendrocytes to immune attack. This conundrum led us to explore the possible contribution of CD4 T cells to enhanced viral control and pathology, especially as CD4 T cells are potent producers of IFN-γ in vivo, and are strongly associated with pathogenicity and clinical disease [4, 24].
The current study characterizes the relative contributions of CD4 T-cell help and B7-H1 to CD8 T-cell effector function in the CNS during viral encephalomyelitis by addressing three interrelated questions. 1) Is enhanced CD8 effector T-cell activity and viral control in B7-H1−/− mice dependent on local CD4 T cells, similar to WT mice? 2) Is CNS CD4 T-cell function dampened by B7-H1? 3) Is increased axonal damage in the absence of B7-H1 sustained in the absence of CD4 T cells? To ensure presence of CD4 T-cell help during priming but withdrawal of helper function within the CNS, CD4 T cells were depleted in B7-H1−/− mice subsequent to the initial expansion phase, but before CNS entry . Numbers and composition of CNS-infiltrating cells were comparable in CD4-depleted mice; however, decreased granzyme B and IFN-γ expression by CD8 T cells correlated with loss of viral control. Moreover increased CD8 function was associated with increased CD4 T-cell activity in B7-H1−/− mice. The absence of CD4 T cells in B7-H1−/− mice did not affect demyelination or significantly lessen axonal damage, but did improve clinical disease and survival.
All procedures were conducted in accordance with animal protocols approved by the Institutional Animal Care and Use Committee.
Mice, virus infection, and CD4 depletion
The WT mouse strain was C57BL/6 (National Cancer Institute (Frederick, MD, USA). B7-H1−/− mice on a C57BL/6 background were previously described . Mice were housed under pathogen-free conditions at an accredited facility in the Cleveland Clinic Lerner Research Institute. Mice were infected at 6–7 weeks of age by intracranial injection with 250 plaque-forming units (PFUs) of the gliatropic JHM variant V2.2-1 of mouse hepatitis virus (JHMV) . Recipient animals were depleted of CD4 T cells by intraperitoneal injection with 250 μg of anti-CD4 (α-CD4) monoclonal antibody (mAb) GK1.5 at 4 and 6 days post-infection (p.i.). Control animals received the same amount of α-βgalactosidase (α-βgal) control mAb GL113. Recipients were depleted of CD8 T cells by intraperitoneal injection with 250 μg of anti-CD8 (α-CD8) mAb 2.43 at day −2, 0, and 7 p.i. These regimens resulted in more than 99% depletion of CD4 or CD8 T cells in the periphery and CNS. Control animals received the same amount of control mAb GL113. Animals were scored daily for clinical signs of disease on a four-point scale (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; 4 = moribund or dead).
Virus titers and cytokine determination
Virus titers within the brain were determined in clarified supernatants by plaque assay, using the murine DBT astrocytoma cell line as described previously . Plaques were counted 48 hours after incubation at 37°C. Clarified supernatants were also used to measure IFN-γ by ELISA as described . Briefly, 96 well plates were coated overnight at 4°C with 100 μl of 1 μg/ml of α-IFN-γ (R4-6A2; BD Biosciences, San Jose, CA, USA). Non-specific binding was blocked with 10% fetal calf serum in PBS for 1.5 h before the addition of IFN-γ recombinant cytokine standard (BD Biosciences) and samples. After a 2 h incubation at room temperature bound IFN-γ was detected using biotinylated α-IFN-γ antibody (XMG1.2, BD Biosciences) and avidin peroxidase followed by 3,3′,5,5′ tetramethylbenzidine (TMB Reagent Set; BD Biosciences) 1h later. Optical densities were read at 450 nm in a microplate reader (Model 680; Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using Microplate Manager software (version 5.2; Bio-Rad Laboratories).
Isolation of mononuclear cells
CNS-derived cells were isolated as described previously . Briefly, brains or spinal cords from PBS-perfused mice (n = 3 to 6) were homogenized in ice-cold Tenbroeck tissue grinders in Dulbecco’s PBS. Homogenates were clarified by centrifugation at 400 g for 7 minutes, and the supernatants were collected and stored at −80°C for further analysis. Cell pellets were resuspended in RPMI supplemented with 25 mmol/l HEPES, adjusted to 30% Percoll (Pharmacia, Piscataway, NJ, USA) and underlaid with 1 ml of 70% Percoll. After centrifugation at 800 g for 30 minutes at 4°C, cells were recovered from the 30/70% interface, washed once, and resuspended in fluorescence-activated cell sorting (FACS) buffer. CNS-derived cell populations for PCR analysis were isolated from infected mice as described above. Cell suspensions from cervical lymph nodes (CLNs) were prepared from identical animals as previously described .
Flow-cytometry analysis and fluorescence-activated cell sorting
Cells were incubated with mouse serum and rat α-mouse FcγIII/II mAb for 15 minutes on ice before staining. Expressionof cell surface markers was determined by incubation of cells with fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated, Peridinin Chlorophyll Protein Complex (PerCP) (PerCP)-conjugated, or allophycocyanin-conjugated mAbs specific for CD45 (30-F11), CD4 (L3T4), CD8 (53–6.7) CD44 (IM7), CD62L (MEL-14) (all BD Biosciences), PD-1 (RMP1-30; eBioScience San Diego, CA, USA) and F4/80 (CI:A3-1; Serotec, Raleigh, NC, USA) for 30 minutes on ice. Virus-specific CD8 T cells were identified using Db/S510 MHC class I tetramers (Beckman Coulter Inc., Fullerton, CA, USA) as described previously . Stained cells were washed twice with FACS buffer and fixed in 2% paraformaldehyde. For intracellular detection of granzyme B or IFN-γ, the cells were stained for cell surface markers before permeabilization (Cytofix/Cytoperm Reagent; BD Biosciences) and staining with allophycocyanin-labeled α-granzyme B Ab (GB12, isotype-control mouse IgG1; Caltag Laboratories Burlingame, CA, USA) or α-IFN-γ Ab (BD Biosciences). A minimum of 2 × 105 viable cells were stained and analyzed on a flow cytometer (FACS Calibur; BD, Mountain View, CA, USA). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). CNS monocyte-derived CD45hiF4/80+ macrophages, CD45lo microglia, and CD4 and CD8 T cells were purified from pooled brains (n = 6 to 8) using a cell sorter (FACSAria; BD). CD4CD44hiCD62Llo (effector) and CD4CD44loCD62Lhi (naive) cells were also purified from pooled CLNs. A minimum of 5 × 104 cells were collected per pooled sample, and frozen in 400 μl Trizol reagent (Invitrogen, Carsbad, CA, USA) at −80°C for subsequent RNA extraction and PCR analysis as described previously .
Virus-specific IFN-γ production by CLN-derived CD8 T cells was evaluated after peptide stimulation. Briefly, 2 × 106 CLN cells were cultured in the absence or presence of 1 μmol/l S510 peptide encompassing the H-2Db-restricted CD8 T-cell epitope in a total volume of 200 μl RPMI supplemented with 10% fetal calf serum for 5h at 37°C with a protein transport inhibitor (GolgiStop; BD Bioscience) at 1 μl/ml. After stimulation, cells were stained for surface expression of CD8, CD44, and CD62L, fixed, and then permeabilized to detect intracellular IFN-γ as recommended by the supplier (BD Biosciences).
Spinal cords from PBS-perfused mice were fixed in 10% formalin and embedded in paraffin. In some experiments, the spinal cords were sectioned longitudinally, while in others they were cut into six segments from cervical to lumbar regions, and embedded together in paraffin. Cross-sections from individual mice were examined at each of the six levels. Demyelination was determined by staining 5 μm sections with Luxol fast blue (LFB), while axonal integrity was examined using the α-phosphorylated neurofilament mAb SMI-31 and the α-non-phosphorylated neurofilament mAb SMI-32 (Covance, Princeton, NJ). Viral nucleocapsid protein was detected by immunoperoxidase staining using the α-JHMV mouse mAb J.3.3 as the primary antibody, horse α-mouse as secondary antibody and 3,3′-diaminobenzidine (DAB) as substrate (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). Microglia and infiltrating macrophages were identified by immunoperoxidase staining using α-Mac-3 (BD Bioscience) as the primary antibody and rabbit α-rat as secondary antibody. With the exception of the LFB staining, all sections were counterstained with hematoxylin. Double immunoperoxidase staining for an oligodendroglial marker (rabbit α-glutathione S-transferase (α-GST; Enzo Life Sciences, Farmingdale, NY, USA) and viral antigen (J.3.3 mAb) was performed on paraffin embedded sections after antigen retrieval (Vector Laboratories);. Immunolabeling was identified using a commercial kit (Vectastain ABC) with DAB and Vector SG chromogens, respectively.
High-resolution digital images were obtained using a slide scanner (ScanScope; Aperio, Vista, CA, USA) with a 20× lens objective and doubling lens. Images were viewed using ImageScope software (Aperio). For semi-quantitative analyses, sections were scored in a blinded fashion, and representative fields were identified based on the average score of all sections in each experimental group. For quantitative analyses (for example, Mac-3 and SMI-31 plus SMI-32 immunolocalization) images were analyzed using the ‘positive pixel count v9’ algorithm (Aperio) to obtain the percentage of positive pixels in representative fields of 5 mm2.
where CT represents the threshold cycle at which the fluorescent signal becomes significantly higher than that of the background.
Results are expressed as the mean ± SEM for each group of mice. In all cases, P<0.05 was considered significant. Graphs were plotted and statistics assessed using GraphPad Prism software (version 3.0).
B7-H1 deficiency does not compensate for the essential anti-viral role of CD4 T-cell help
Absence of B7-H1 does not rescue anti-viral function of unhelped CD8 T cells
B7-H1 regulates CD4 T-cell function in the CNS
CD4 T cells are mediators of exacerbated disease in B7-H1−/−mice
Control of viral CNS infections is dependent on CD8 T-cell effector function in numerous rodent models [19, 41–44]. Because many neurotropic viruses infect resident cells capable of upregulating MHC class I, but not class II molecules, effector mechanism are thought to result from direct target-cell/T-cell interactions. However, CD4 T cells play a supportive role for CD8 T cells that is less well understood, especially given the limited MHC class II expression, restricted to infiltrating APCs and microglia. Making matters more complex, effector functions by both T-cell subsets are regulated by inhibitory interactions in a cell type-specific manner. Specifically, B7-H1 blockade or deficiency enhances or reinvigorates CD8 T-cell function during prolonged antigen exposure in both visceral tissues and the CNS [3, 45–48]. The extent to which potentially exacerbated CD4 T-cell function in the absence of B7-H1 inhibition influences CD8 T-cell activity and viral control is poorly explored. During gliatropic JHMV infection, CD8 T cells play a dominant role in controlling virus replication, but robust IFN-γ-mediated B7-H1 upregulation on oligodendrocytes, the prominent target of infection, delays viral control and contributes to persistence [15, 22]. Accelerated viral control in the absence of B7-H1 thus provides a model to test whether CD4 T-cell help promoting CD8 T-cell activity in the CNS of WT mice is also a driving force for enhanced CD8 T cell activity in B7-H1−/− mice.
The results clearly indicate that CD4 T cells are crucial in promoting the enhanced anti-viral CD8 T-cell response in B7-H1−/− mice. Despite reduced CNS chemokine expression, likely resulting from reduced IFN-γ (data not shown), the absence of CD4 T cells did not affect initial CNS accumulation of CD8 T cells in B7-H1−/− mice, similar to WT mice . Reduced expression of perforin and IFN-γ transcripts and granzyme B by CNS-derived B7-H1−/− unhelped CD8 T cells coincided with loss of CNS viral control at day 10 p.i. The sustained viral loads are likely responsible for the increased CNS numbers of total CD8 T cells at day 14 p.i. relative to control mice. Nevertheless, CD4-depleted B7-H1−/− mice harbored at least 10-fold lower viral titers at days 10 and 14 p.i. relative to CD4-depleted WT mice, supporting higher activity of B7-H1−/− CD8 T cells compared with WT CD8 T cells, regardless of CD4 help. This was substantiated by higher granzyme B levels in unhelped B7-H1−/− CD8 T cells relative to unhelped WT CD8 T cells, and provides evidence that B7-H1 deficiency also directly contributed to enhanced CNS CD8 T-cell activity in the absence of CD4 T cells.
A direct inhibitory effect of B7-H1 engagement by CD4 T cells was suggested by the high expression of PD-1 on CNS-infiltrating CD4 T cells during JHMV infection in WT mice. B7-H1 mediated inhibition was indeed supported by increased IFN-γ, IL-21 and IL-10 transcript levels in CNS-derived B7-H1−/− CD4 T cells relative to their WT counterparts. Increased CD4 T cell activity in the CNS of B7-H1−/− mice has also been reported in the experimental allergic encephalomyelitis (EAE) model  and may indirectly contribute to increased anti-viral CD8 T-cell activity in the absence of B7-H1 in the CNS. Although increased IFN-γ and IL-10 mRNA in B7-H1−/− relative to WT CD4 T cells was already imprinted during priming in CLNs, enhanced local CNS restimulation was supported by the specific increase of IL-21 transcripts and the overall 5-fold to 10-fold higher IFN-γ and IL-10 transcript levels in the CNS relative to CLN-derived B7-H1−/− CD4 T-cell populations. Potential APCs which mediate B7-H1 suppressors of CD4 T cells within the JHMV-infected CNS are MHC class II positive macrophages, based on their strong B7-H1 expression compared with the modest and transient expression on microglia . Although only few microglia/macrophages are infected, recent evidence demonstrates that CD4 T cells require only a few MHC class II-presenting cells to elicit IFN-γ secretion and long-range responsiveness in vivo. However, although direct in vivo evidence for DC infection has been elusive, it cannot be excluded that viral antigen cross-presentation by DCs contributes to enhanced B7-H1−/− CD4 T-cell effector function in the CNS. DCs presumably initiate T-cell priming within the CLNs, and the absence of constitutive B7-H1 expression on these APCs gives rise to enhanced activation of CLN-derived B7-H1−/− CD4 T cells. Expression of IFN-γ and IL-10 mRNA was also greater in CLN-derived B7-H1−/− CD8 T cells compared with their WT counterparts, although these differences were not reflected in the expansion of virus-specific CD8 T cells (data not shown). Overall, these results support early imprinting of B7-H1 on both CD8 and CD4 T-cell function during priming, which are further amplified by T cell restimulation within the CNS.
In addition to revealing a pronounced direct effect of B7-H1 on dampening CD4 T-cell activity, CD4 T-cell depletion indicated these lymphocytes as primary mediators of exacerbated disease in B7-H1−/− mice, independent of increased viral load. This was supported by the inability of CD8 T-cell depletion to ameliorate disease in B7-H1−/− mice. The implication that CD8 T cells alone might be candidates causing exacerbated disease ,based on more rapid viral control in B7-H1−/− oligodendrocytes , which express MHC class I, but not class II, was thus not sustainable. Furthermore, whereas CD4 depletion in B7-H1−/− mice ameliorated disease, despite impairing viral control, improvement of axonal damage did not reach statistical significance. These data, combined with similar demyelination, suggested additional affects of over-activated CD4 T cells on neuronal function, not necessarily reflected in histological readouts of neuronal integrity. Nevertheless, preservation of neurological function also correlates with axon sparing, regardless of demyelination during infection with Theiler’s murine encephalomyelitis virus [51, 52]. The mechanisms underlying neuronal damage may be to due to misdirected T-cell activity or to secondary bystander effects. The absence of enhanced neuronal infection in CD4-competent B7-H1−/− mice excluded a direct virus-mediated effect. Significantly decreased IFN-γ, reduced iNOS mRNA expression, and reduced Mac-3 reactivity in the CNS of CD4-depleted B7-H1−/− mice supports a direct contribution of CD4-mediated microglia/macrophage activation to disease severity and mortality. Although macrophages can mediate demyelination in the absence of T cells during JHMV infection , apparent preservation of axonal integrity under these conditions suggests that CD4-mediated activation of the macrophages promotes axonal dysfunction. Involvement of blood-derived infiltrating macrophages in JHMV pathogenesis is also supported by decreased clinical severity in CCL2−/− mice . Similarly, inhibition of macrophage function in other experimental CNS diseases, such as EAE and encephalomyelitis caused by Theiler’s murine encephalomyelitis virus, ameliorates disease [55–57]. A contribution of iNOS as a detrimental effector molecule is consistent with the association between increased levels of iNOS expression and the pathological changes in other CNS disorders such as multiple sclerosis and its animal correlate EAE [58–62]. iNOS synthesizes the free radical nitric oxide (NO), which is implicated in pathologic processes due to its cytotoxicity at high concentrations and the destructive molecules generated from NO such as peroxynitrite [63–66] . Although iNOS does not seem to contribute directly to JHMV pathogenesis , it cannot be excluded that increased levels of NO or its products in B7-H1−/− mice have deleterious local effects in the CNS.
This study demonstrates that CD4 T-cell helper functions contribute significantly to enhanced CD8 T-cell activity in B7-H1−/− mice, in addition to the direct relief of CD8 T cells from PD-1:B7-H1 inhibitory signaling within the CNS. PD-1:B7-H1 blockade is thus insufficient to overcome CD4 T cell helper function to CD8 T cells during JHMV infection. Moreover, CD4 T cells are prominent contributors to bystander pathology during JHMV infection if not regulated by B7-H1:PD-1 interactions.
Cervical lymph node
Central nervous system
Fluorescence-activated cell sorting
glyceraldehyde 3-phosphate dehydrogenase
Inducible nitric oxide synthase
Gliatropic JHM strain of mouse hepatitis virus
Luxol fast blue
Major histocompatibility complex
Peridinin chlorophyll protein complex
This work was supported by National Institutes of Health grants NS064932 and AI47249. We sincerely thank Wenqiang Wei and Eric Barron for their exceptional technical assistance, and Jennifer Powers for FACS purification.
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