Differential expression of B7 co-stimulatory molecules by astrocytes correlates with T cell activation and cytokine production

Jeanne M. Soos, Timothy A. Ashley, Jennifer Morrow, Juan Carlos Patarroyo2, Brian E. Szente1 and Scott S. Zamvil2

Center for Neurologic Diseases, Harvard Institutes of Medicine, and
1 Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA
2 Department of Neurology, C-440, University of California, San Francisco, 521 Parnassus Avenue, San Francisco, CA 94143-0114, USA

Correspondence to: S. S. Zamvil as above; Email: zamvil{at}itsa.ucsf.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Whether astrocytes utilize B7:CD28 co-stimulation to activate T cells mediating CNS inflammatory disease is controversial. In this report, primary astrocytes and murine astrocyte lines, generated by immortalization at two different times, day 7 or 45 of culture, were examined for their capability to express B7 co-stimulatory molecules and to participate in B7:CD28 co-stimulation. Following exposure to IFN-{gamma}, primary astrocytes and astrocyte lines up-regulated MHC class II and B7-2 (CD86) molecules. However, B7-1 (CD80) expression was not inducible on primary astrocytes examined after IFN-{gamma} stimulation beginning on day 7 or on astrocyte lines immortalized on day 7. B7-1 expression was inducible on primary astrocytes examined later and could be up-regulated on astrocyte lines immortalized later. Unlike B7-1, temporal discordant expression of other co-stimulatory/adhesion molecules was not observed. Both B7-1/B7-2+ and B7-1+/B7-2+ astrocyte lines were capable of stimulating proliferation of encephalitogenic Th1 cells, utilizing B7-2 for B7:CD28 co-stimulation. However, lines derived from immortalization later (B7-1+/B7-2+) were more effective in stimulating proliferation of naive myelin basic protein-specific CD4+ T cells. Astrocyte lines that expressed both B7-1 and B7-2 also stimulated Thp cells to secrete proinflammatory Th1 cytokines, whereas lines that expressed B7-2 only stimulated Thp cells to produce a Th2 cytokine pattern. Thus, we demonstrate for the first time that individual astrocytes can differentially express B7-1 molecules, which may correlate with their ability to stimulate proinflammatory and regulatory patterns of cytokine production. These results suggest that astrocytes have potential for both promoting and down-regulating T cell responses, and that temporal differences in expression of B7 molecules should be considered when evaluating immune regulation by astrocytes.

Keywords: antigen presentation, astrocytes, costimulation, experimental allergic encephalomyelitis, MHC class II, multiple sclerosis, T cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Whether astrocytes, the most abundant glial cell population within the CNS, participate in the activation of T cells causing CNS inflammatory disease is not clear. It has been reported that astrocytes within inflammatory lesions of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE) express MHC class II molecules, suggesting that they may serve as antigen-presenting cells (APC) for the activation of CD4+ T cells in vivo (1,2). In vitro studies have shown that astrocytes exposed to IFN-{gamma} up-regulate class II molecules and can present antigen to T cells (38). It has recently been reported that IFN-{gamma}-activated astrocytes can also express elements of the endocytic pathway (8,9) and are capable of processing native CNS autoantigen (9), a function that could be necessary for initiating activation of autopathogenic T cells in vivo. However, certain reports indicate that astrocytes are less competent APC (5,1012), and suggest that astrocytes may serve to down-regulate CNS inflammatory responses (13,14). It is also not clear whether astrocytes are capable of activating unprimed T cells (5), which could be relevant to the recruitment of T cells involved in the later phases of CNS inflammatory responses (15).

B7-1 (CD80) and B7-2 (CD86) are cell surface proteins on APC that provide potent co-stimulatory signals for T activation and differentiation through their interaction with the T cell counter-receptor, CD28 (16,17). Results from several investigations in EAE (1821), as well as other T cell-mediated inflammatory and autoimmune conditions (2226), have indicated that B7-1 and B7-2 have distinct effects in the differentiation of precursor T cells (Thp) into proinflammatory (Th1) and regulatory (Th2) cells. In vivo studies of EAE indicate that B7-1 promotes differentiation of encephalitogenic Th1 cells (1820) and some studies also suggest that B7-2 promotes Th2 differentiation (18,20). Although the role of B7:CD28 co-stimulation in T cell activation and regulation by individual resident CNS accessory cell subpopulations has been investigated in both MS and EAE (8,2729), the relative contribution of B7-1 and B7-2 expression to T cell activation within the CNS has not yet been clarified. In examination of MS brain, some investigators reported that B7-1 molecules were expressed primarily on microglia (27,28) and not astrocytes (28). However, in one recent study of relapsing EAE, B7-2 co-stimulatory molecules were observed on both astrocytes and microglia, while B7-1 expression on astrocytes occurred later (29). Reports regarding B7 expression and B7:CD28 co-stimulation by astrocytes examined in vitro have also conflicted (7,8,11,12,28,30). Some investigators have reported that IFN-{gamma}-stimulated astrocytes express both B7-1 and B7-2 molecules, and participate in B7:CD28 co-stimulation through the engagement of B7-2, but not B7-1, molecules with CD28 expressed on T cells (7). In contrast, other investigators have observed that astrocytes do not express B7-1 or B7-2 co-stimulatory molecules and do not utilize the B7:CD28 co-stimulatory pathway (11,12,30).

In the present investigation we have examined whether astrocytes can differentially express B7-1 and B7-2 costimulatory molecules for B7:CD28 co-stimulation, a possibility that could influence their capability to serve as APC in T cell activation and regulation, and could also provide an explanation for the conflicting results reported regarding T cell activation by astrocytes (7,8,11,12,28,30). In order to examine this possibility, immortalized and primary astrocytes were examined at two different time periods. Immortalization can capture cells at different stages of development, allowing one to examine whether there are temporal changes during differentiation. In addition, immortalization allows one to select pure astrocyte lines, which can be examined in the absence of potential contaminating microglia, a concern raised by other investigators who have studied B7 expression and B7:CD28 co-stimulation by astrocytes (7,12). We have observed that astrocytes immortalized earlier express B7-2, but not B7-1, molecules even when activated by IFN-{gamma}, while astrocytes immortalized later in culture up-regulated both B7-1 and B7-2 molecules following IFN-{gamma}-stimulation. Similar to the immortalized astrocytes, expression of B7-1 molecules on primary astrocytes was not observed earlier, but was inducible by IFN-{gamma} stimulation later. We observed that B7-2 expression by astrocytes is sufficient for the activation of mature encephalitogenic T cells, suggesting that B7-2 expression by astrocytes could contribute to the activation of encephalitogenic T cells in the acute phase of EAE. Although B7-1/B7-2+ astrocytes did not support proliferation of Thp, they induced Thp to produce a Th2 cytokine pattern. In contrast, expression of B7-1 by astrocytes correlated both with more effective activation of Thp cells and the secretion of Th1 cytokines, suggesting that some astrocytes have potential to activate naive T cells to differentiate into proinflammatory Th1 cells. Thus, the potential for temporal changes in B7-1 expression should be considered when investigating T cell activation by primary astrocytes. Differential expression of B7-1 co-stimulatory molecules may influence the ability of astrocytes to serve as functional APC for the stimulation of T cells in vivo.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Development of primary astrocyte cultures
Primary glial cultures were prepared from the cerebral hemispheres of newborn B10.PL (H-2u) mice using established methods (31) as described previously (9). After removal of the meninges under a dissecting microscope, tissue from the cerebral lobes was dissociated and passed through a sterile 70 µm nylon cell strainer. This cell suspension was centrifuged, then treated with trypsin–EDTA for 5 min at 37°C. Cells separated in this manner were then washed and plated in complete RPMI containing 10% heat-inactivated FCS, L-glutamine and penicillin/streptomycin. Cells were initially plated at 5x104 cells/cm2 for enrichment of astrocyte growth or grown in 75 cm2 flasks that had been treated for 1 h with poly-D-lysine (10 mg/ml) and washed 3 times with sterile water. Cells plated in flasks were cultured for 7 days. In order to remove non-adherent cells, including oligodendrocytes and microglia, flasks were agitated for 8 h at 200 r.p.m. on an orbital shaker. Media, containing non-adherent cells, was removed. Media was replaced and agitation was continued for an additional 18 h at 200 r.p.m. Again, non-adherent cells were removed and media replaced. Ninety-seven percent of the remaining cells were GFAP+ and <1% stained positive for Mac-1, a cell surface molecule expressed on microglia, monocytes and macrophages.

Immortalization of primary astrocytes
The SV40-406 cell line produces a replication defective retrovirus encoding the SV40 large T antigen in the pZipNeoSV(X)1 construct (32,33) containing neomycin phosphotransferase (Neo). Immortalization by this method can `fix' cells in differentiation, but does not confer tumorigenicity (32). SV40-406 cells were plated at 5x105 cells/75 mm2 plate in 10 ml of RPMI containing 10% heat-inactivated FCS and grown for 24 h (9). The media was changed, then collected 24–48 h later and stored at –80°C. Astrocytes cultured in vitro were exposed to SV40-406 culture supernatants for 2 h in the presence of polybrene (4 µg/ml). After 1 day or more of treatment, cells were exposed to G418 (400 µg/ml). Astrocyte lines were obtained from immortalization of three separate primary cultures. Three colonies were recovered from cells immortalized after 7 days, providing lines 3.1, 3.2 and 3.3. Two colonies were obtained from cells immortalized after 45 days in culture, providing lines 1.1 and 2.1 from two separate primary cultures. Each colony was recovered from a separate plate and no more than one colony was observed on each plate. Thus, each line was the result of a separate transformation event. All astrocyte lines contain GFAP, an intermediate filament protein expressed primarily by astrocytes (data not shown). They do not express Mac-1, a cell-surface antigen expressed on microglia and macrophages, or galactocerebroside, a marker for oligodendrocytes. These independent astrocyte lines have been maintained for >1 year.

Antigens
Myelin basic protein (MBP) peptide Ac1–11 (ASQKRPSQRHG) was synthesized by solid-phase Fmoc chemistry by Quality Control Biochemicals (Hopkinton, MA). After cleavage from the solid support and deprotection of the amino acid side chains, peptides were purified by reversed-phase HPLC (C18 column, YMC). Major peaks were analyzed by MALDI-TOF mass spectrometry and HPLC. Each peptide contained >95% of the desired product. Native murine, bovine and human MBP were purified as described (34).

MBP-specific T cells
PJR-25 is an encephalitogenic T cell clone derived from the PL/J mouse strain that is specific for MBP Ac1–11 in association with I-Au (35). PJR-25 proliferates in response to intact mouse, bovine, guinea pig and human MBP (36). `Naive' splenic CD4+ T cells were purified from transgenic mice bearing the TCR specific for MBP Ac1–11 in association with I-Au using CD4 purification columns (Cytovax, Edmonton, Canada).

mAb
The following mAb were used in this study: AF6-120.1 (murine anti-I-Ab,u)–FITC, M1/70 (rat anti-mouse Mac-1), 1G10 [rat anti-mouse B7-1 (CD80)] and GL1 [rat anti-mouse B7-2 (CD86)] were obtained from PharMingen (San Diego, CA). Rabbit anti-bovine GFAP (cross-reactivity to mouse) was obtained from Dako (Carpinteria, CA). Anti-galactocerebroside was obtained from Sigma (St Louis, MO). FITC-conjugated isotype-matched or secondary antibodies were all obtained from PharMingen.

Proliferation assays
To measure antigen-specific T cell proliferation, APC (astrocytes and splenocytes) were first treated with mitomycin C (60 µg/ml/106 APC) for 1 h at 37°C and washed 3 times with media. T cells (104/well), either the T cell clone PJR-25 or CD4+ purified T cells from MBP-TCR transgenic mice, were cultured in 96-well plates with the MBP peptide Ac1–11 at various concentrations and APC (4x104/well). Media used for the proliferation assays was RPMI supplemented with 5% heat-inactivated FCS and L-glutamine. In all proliferation experiments, cultures were incubated for 48 h at 37°C. [3H]Thymidine (1 µCi/ml) was added and cultured for 18 h prior to harvesting and counting. The mean c.p.m. of [3H]thymidine incorporation were calculated for triplicate cultures.

Flow cytometry
For staining cell surface molecules, astrocytes cultured alone or treated with IFN-{gamma} (100 U/ml) for 48 h were removed from flasks by treatment with PBS/EDTA, counted, then aliquoted at 106 cells/tube and washed with FACS buffer (PBS containing 0.5% BSA and 10 mM sodium azide). Cells were stained with either primary unlabeled mAb or FITC–mAb for 45 min at room temperature, then washed. In all cases, isotype-matched control unlabeled mAb or FITC–mAb were included as controls. When the primary mAb was unlabeled, cells were then incubated for 45 min with a FITC–antibody directed against the primary mAb, then washed. Samples were analyzed on a FACSort (Becton Dickinson, Mountain View, CA) using 10,000 events per sample.

Staining of intracellular proteins (GFAP and galactocerobroside) was performed as described (37). After washing cells in FACS buffer they were treated with a 2.5% paraformaldehyde solution for 10 min at room temperature, washed in saponin buffer (PBS containing 0.1% saponin and 10 mM sodium azide), then incubated with primary mAb for 1 h at room temperature. Cells were washed with saponin buffer and incubated for 1 h with a FITC–antibody directed against the primary mAb. Again, cells were washed with saponin buffer, then resuspended in FACS buffer and analyzed by FACS as described above.

Cytokine ELISA
Supernatants from proliferation assays were obtained 24–48 h after initiation of culture. Ninety-six-well plates were coated overnight in carbonate buffer containing anti-cytokine capture antibody (1 µg/ml). The plates were washed and blocked for 2 h with 10% blocking solution (Kirkegaard & Perry, Gaithersburg, MD). The plates were washed and supernatants from proliferation assays and the appropriate standards were added and incubated overnight at 4°C. The plates were washed again and the appropriate biotinylated anti-cytokine detecting mAb (1 µg/ml) was added and incubated for 1 h at room temperature. The plates were washed and further developed by addition of avidin–peroxidase (Sigma, St Louis, MO) and substrate. Absorbance was measured at 450 nm. The amount of cytokine in each supernatant was extrapolated from the standard curve. The standards used were recombinant cytokine curves generated using 1:2 dilutions of which the initial concentration used for IL-4 was 1600 pg/ml, IL-2 was 3200 pg/ml and IFN-{gamma} was 6400 pg/ml.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Time-dependent B7 co-stimulatory molecule expression by immortalized astrocyte lines and primary astrocytes
Individual immortalized astrocyte lines were generated from primary B10.PL (H-2u) astrocyte cultures using a replication-defective retrovirus containing SV40 large T antigen construct (32,33) (see Methods). Immortalization of murine astrocytes by this method does not confer tumorigenicity (32). Primary astrocytes were immortalized either after 7 or 45 days in culture. Astrocyte lines generated by immortalization expressed GFAP, but not Mac-1 (data not shown). Low levels of constitutive B7-2 expression were observed on all astrocyte lines, which increased significantly after IFN-{gamma} stimulation (Fig. 1Go). Following IFN-{gamma} stimulation astrocyte lines immortalized at the later time point (1.1 and 2.1) also expressed B7-1. In contrast, when astrocytes immortalized earlier in culture (3.1, 3.2, and 3.3) were examined, neither constitutive nor inducible B7-1 expression was observed. Lack of B7-1 expression was also observed when these astrocytes (3.1, 3.2 and 3.3) were stained with a separate anti-B7-1 mAb (16-10A1), although this second anti-B7-1 mAb, like the one used for B7-1 staining shown in Fig. 1Go, stained positively for B7-1 when staining lines 1.1 and 2.1 (data not shown). Similar to primary astrocytes, these astrocyte lines did not express cell surface class II without stimulation with IFN-{gamma} although class II (I-Au) expression was induced by IFN-{gamma} stimulation (Fig. 1Go) In several separate experiments class II expression by line 3.2 was consistently less than other astrocyte lines. In some experiments (Fig. 1Go) less B7-2 expression was also observed on line 3.2, although in other experiments (data not shown) the levels of B7-2 up-regulation by this line were similar to the other lines when tested at the same time.



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Fig. 1. Expression of B7 co-stimulatory molecules and MHC class II by immortalized astrocytes. Immortalized astrocytes were treated with IFN-{gamma} (100 U/ml) or media for 48 h and then examined by FACS analysis. The bold line represents staining of IFN-{gamma}-treated cells with the relevant mAb, the thin line represents staining of media-treated cells with the relevant mAb and the dotted line represents staining with isotype-matched mAb.

 
Astrocyte lines were also examined for expression of other co-stimulatory/adhesion molecules. Similar to previous observations of primary astrocytes (7,12), constitutive ICAM and VCAM expression was observed on all of the astrocyte lines, regardless of the timing of immortalization (see Fig. 2Go). Up-regulation of ICAM was observed on all astrocyte lines. No significant constitutive or inducible CD40 expression was observed on any of these lines. Thus, in contrast with the appearance of IFN-{gamma}-inducible B7-1 expression on astrocytes immortalized later, temporally discordant expression of these other co-stimulatory/adhesion molecules was not observed.



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Fig. 2. Analysis of ICAM-1, VCAM and CD40 expression by immortalized astrocytes. Data from two representative immortalized astrocyte lines (3.1 (day 7) and 2.1 (day 45)) are shown. In (A) immortalized astrocytes were treated with IFN-{gamma} (100 U/ml) or media for 48 h, and then examined for expression of ICAM and VCAM by FACS analysis. In a separate experiment, immortalized astrocytes treated in the same manner were examined for expression of CD40 (B). In this experiment (B), monocyte-enriched splenocyte cultures were stained in parallel. Splenocyte cultures were enriched for monocytes by plastic adherence and treated with LPS (100 ng/ml) or media for 48 h. In each FACS profile shown, the bold line represents staining of IFN-{gamma}- or LPS-treated cells with the relevant mAb, the thin line represents staining of media-treated cells with the relevant mAb and the dotted line represents staining with isotype-matched mAb.

 
In order to further evaluate whether the differential B7-1 expression observed on immortalized lines reflected a temporal change in B7 expression in a larger astrocyte population, B7-1 and B7-2 expression was examined on primary astrocyte cultures at two different time points (Fig. 3Go). Unactivated astrocytes and astrocytes activated with IFN-{gamma} for 48 hours beginning on day 7 and examined on day 9 showed expression of B7-2 while B7-1 was not detectable. However, when unactivated astrocytes and astrocytes activated with IFN-{gamma} for 48 hours beginning on day 28 were examined on day 30, expression of both B7-1 and B7-2 was observed. Similarly, when examined with a second anti-B7-1 (16-10A1), B7-1 expression was not detected on unstimulated or IFN-{gamma}-stimulated primary astrocytes examined on day 9, but was detected on IFN-{gamma}-stimulated primary astrocytes that were examined on day 30 (data not shown). Thus, B7-1 expression by astrocytes in vitro appears to be influenced by the duration of culture.



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Fig. 3. Time-dependent B7-1 expression on primary astrocytes. Primary astrocytes were activated with IFN-{gamma} for 48 hours beginning on day 7 or day 28. These cultures were then examined on days 9 and 30, respectively, by FACS analysis for expression of B7-1, B7-2, GFAP and Mac-1. For detection of B7 expression, the bold line represents staining of IFN-{gamma}-treated cells with relevant mAb, the thin line represents staining of media-treated cells with the relevant mAb and the dotted line represents staining with isotype-matched mAb. For detection of GFAP, the bold line represents staining with primary and secondary antibodies, and the thin line represents staining with secondary antibody. For detection of Mac-1, the bold line represents biotinylated primary antibody and strepavidin–PE and the thin line represents staining with isotype-matched mAb and strepavidin–PE.

 
Differential B7 expression correlates with capability of individual astrocyte lines to stimulate MBP-specific T cell proliferation
Astrocyte lines were examined for their capability to stimulate both mature and naive MBP-reactive CD4+ T cells. As shown in Table 1Go, four of five IFN-{gamma}-stimulated astrocyte lines could stimulate proliferation of encephalitogenic mature MBP Ac1–11-specific Th1 cells. Only a minimal proliferative response was observed when line 3.2, which expressed the lowest levels of class II molecules, served as APC. As only astrocyte lines 1.1 and 2.1 immortalized at the late time point express B7-1, these results suggest that B7-1 co-stimulation by astrocytes is less critical for stimulating proliferation of mature encephalitogenic Th1 cells.


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Table 1. Proliferative responses by mature and naive MBP-reactive T cells to MBP Ac1–11 presented by immortalized astrocyte lines.
 
Using unstimulated purified CD4+ T cells from MBP (Ac1–11) TCR transgenic mice as a source of naive T cells, it was observed that astrocyte lines immortalized at the later time point which express both B7-1 and B7-2 were more capable of stimulating naive T cells to proliferate in response to MBP Ac1–11 (Table 1Go). Stimulation of mature and naive T cells by representative immortalized astrocytes occurs in an antigen dose-dependent manner (Fig. 4Go). Despite their ability to stimulate proliferation of mature MBP-reactive T cells, astrocyte lines 3.1 and 3.3, immortalized at the early time point, which do not express B7-1, were less capable of stimulating proliferation of naive CD4+ MBP Ac1–11-specific T cells. The relative lack of proliferation by these naive CD4+ T cells was not due to anergy or T cell death, as T cells that had been cultured in the presence of either astrocyte line 3.1 or 3.3 proliferated in response to MBP Ac1–11 upon re-stimulation when B10.PL splenocytes were provided as APC (Fig. 5Go).



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Fig. 4. Dose-dependent proliferative responses of mature and naive MBP-specific T cells to antigen presented by representative immortalized astrocytes. Immortalized astrocytes were activated with IFN-{gamma} (100 U/ml) (•) or media ({blacksquare}) alone for 48 h prior to use in assays, treated with mitomycin C, then plated at 4x104 cells/well. In parallel, B10.PL splenocytes were treated with mutomycin C. Mature encephalitogenic MBP Aci1–11-specific T cells (1x104/well) from the PJR-25 clone and purified CD4+ T cells (1x104/ well) from MBP Ac1–11-specific TCR transgenic mice were used as the source of naive T cells.

 


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Fig. 5. Lack of stimulation by immortalized astrocytes 3.1 and 3.3 does not result in T cell anergy. Purified CD4+ T cells (1x104/well) from MBP-TCR transgenic mice were incubated with various concentrations of the MBP peptide Ac1–11 (50 µg/ml is shown) and mitomycin C-treated immortalized IFN-{gamma}-stimulated astrocytes (4x104/well) or B10.PL splenocytes (SP) (4x104/well) respectively. At day 2, a portion of the assay was examined for proliferation (labeled 1° stimulation). The remainder of the assay was then washed and replated in the presence of B10.PL splenocytes (SP) (labeled 2° stimulation). These cultures were then incubated for 2 days with MBP Ac1–11 and harvested to determine proliferation.

 
Whether B7 co-stimulation is important for T cell activation by superantigen remains unclear (3840). It has been shown that exposure to superantigen can alter the course of EAE (4144). However, in one study it was shown that astrocytes do not support T cell activation by superantigen (45). As the murine astrocytes examined in this study express B7 co-stimulatory molecules, we examined whether they could support T cell activation by superantigen. In contrast to B10.PL splenocytes that can activate mature encephalitogenic Vß8.2+ T cells with the Vß8-specific superantigen, staphylococcal enterotoxin B (SEB), none of these IFN-{gamma}-activated class II+ astrocyte lines that expressed B7-2 only or both B7-1 and B7-2 could support activation of encephalitogenic Vß8.2+ T cells by SEB (Fig. 6Go). Thus, the lack of superantigen-mediated T cell activation by astrocytes is not due to a lack of B7 expression.



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Fig. 6. Immortalized astrocyte lines do not support staphylococcal enterotoxin B (SEB)-induced activation of encephalitogenic TCR Vß8.2+ T cells. PJR-25 T cells (Vß8.2+) (1x104) cultured with individual IFN-{gamma}-stimulated astrocyte lines (4x104 cells/well) were treated with SEB (0.5 µg/ml) or with media and proliferation was determined as described in Methods.

 
Differential requirement for B7 co-stimulation for activation of mature and naive MBP-specific T cells by astrocyte lines
The observation that astrocyte lines that did not express B7-1 could stimulate proliferation of mature encephalitogenic MBP Ac1–11-specific T cells suggested that B7-2 provides sufficient B7 co-stimulatory signal for activation of these mature T cells. Consistent with this observation, anti-B7-2 mAb caused a reduction in MBP Ac1–11-specific proliferative responses when either astrocyte lines that expressed B7-2 only or both B7-1 and B7-2 were provided as the source of APC (Fig. 7A and BGo). Anti-B7-1 mAb did not inhibit proliferation, and the combination of anti-B7-1 and anti-B7-2 mAb did not reduce proliferative responses more than anti-B7-2 mAb alone, indicating that that B7-2 provides the predominant B7:CD28 co-stimulatory signal for activation of these mature T cells by astrocytes.



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Fig. 7. Astrocyte lines utilize B7 co-stimulation for activation of mature and naive MBP-specific T cells. (A) Astrocyte line 3.1 (B7-1/B7-2+) utilizes B7-2 co-stimulation for activation of mature MBP-specific T cells. (B) Astrocyte line 2.1 (B7-1+/B7-2+) uses B7-2 co-stimulation for activation of mature MBP-specific T cells. (C) B7-1 and B7-2 co-stimulatory molecules provide co-stimulation for activation of naive MBP-reactive T cells by astrocyte line 2.1. Prior to culture with T cells, the immortalized astrocytes were activated with IFN-{gamma} (100 U/ml) for 48 h, treated with mitomycin C, then plated at 4x104/well. Clone PJR-25 (1x104/well) was used as a source of mature encephalitogenic MBP Ac1–11-specific T cells (A and B). Purified CD4+ T cells (1x104/well) from MBP Ac1–11-specific TCR transgenic mice were used as the source of naive T cells (C). MBP Ac1–11 (50 µg/ml) was provided as a source of antigen. Percent control proliferation ([3H]thymidine incorporation in c.p.m.) was calculated using the mean proliferation from cultures containing antigen (MBP Ac1–11), IFN-{gamma}-activated astrocytes (APC) and T cells (i.e. cultures without addition of any mAb) as the reference. Mean proliferative responses for these controls (100%) were: (A) 62,864, (B) 43,538 and (C) 5164 c.p.m.

 
Astrocyte lines immortalized at the later time point that expressed both B7-1 and B7-2 (1.1 and 2.1) could stimulate proliferation of naive MBP-reactive T cells more efficiently. This observation suggests that either B7-1 alone or the combination of B7-1 and B7-2 may be required co-stimulatory signals for the activation of naive MBP-reactive T cells. As shown in Fig. 7Go(C), when an astrocyte line that expresses both B7-1 and B7-2 was used as APC, exposure to mAb specific for either B7-1 or B7-2 caused a modest reduction in proliferation of naive CD4+ MBP-specific T cells. Although anti-B7-1 and anti-B7-2 inhibition was modest, in all experiments anti-B7-1 mAb reduced proliferation to a greater extent than did anti-B7-2 mAb. However, when anti-B7-1 and anti-B7-2 mAb were used in combination, T cell proliferation was reduced >90%.

Cytokine production by naive MBP-specific T cells correlates with B7 expression on astrocytes
Previous studies indicate that differential B7-1 expression may influence Thp differentiation (1820,2225). Thus, we examined whether the differential expression of B7-1 co-stimulatory molecules by astrocytes influenced Thp differentiation. As shown in Fig. 8Go, when astrocyte lines that express both B7-1 and B7-2 were provided as APC, naive MBP-specific CD4+ T cells secreted significant levels of IFN-{gamma} and IL-2 but less IL-4. In contrast, when the APC were astrocyte lines that expressed only B7-2 (lines 3.1 and 3.3), naive T cells produced more IL-4, very little IFN-{gamma} and no detectable IL-2. The observation that astrocyte lines 3.1 and 3.3 could induce cytokine production, but not proliferation, suggests there is a different threshold for cytokine production and proliferation by these T cells (46). These results are also consistent with other studies which suggest that B7 expression has distinct effects on Thp differentiation (18,19,22,25).



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Fig. 8. Differential cytokine production by naive MBP Ac1–11-specific CD4+ T stimulated by astrocyte lines. Production of IFN-{gamma} (A), IL-4 (B) and IL-2 (C) were examined by ELISA. Purified naive CD4+ T cells (1x104/well) from MBP Ac1–11-specific TCR transgenic mice were stimulated with the MBP peptide Ac1–11 (50 µg/ml) with B7-1 lines (3.1 and 3.3) and B7-1+ lines (1.1 and 2.1). Immortalized astrocytes (4x104/well) were treated with IFN-{gamma} (100 U/ml) or media alone for 48 h prior to use in culture. Supernatants were harvested at 24 or 48 h.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Astrocytes, cerebrovascular endothelial cells (CVE) and microglia are resident CNS accessory cell populations that may participate in antigen-specific T cell activation in acute and chronic CNS inflammatory disease (3,5,10,47,48). While it has been demonstrated that microglia can participate in T cell activation in CNS inflammation (49,50), the potential role of astrocytes, the most abundant resident CNS accessory cell subpopulation, and CVE in T cell activation is less clear (10,48). It has been established that IFN-{gamma}-stimulated primary astrocytes can process native CNS autoantigen, a function that may be necessary for initiating T cell activation in vivo, and that IFN-{gamma}-stimulated astrocytes are capable of activating both mature and naive CD4+ T cells (37,12). However, results from several other investigations indicate that astrocytes are not efficient in initiating primary T cell responses (5,1012). B7:CD28 co-stimulation by CNS APC is thought to be necessary for both the activation of encephalitogenic Th1 cells involved in acute EAE and the recruitment of additional T cells that may participate in the chronic phases of disease (15). In this regard, different groups of investigators have examined B7 expression on astrocytes (7,8,11,12,28,30). Results from these studies have conflicted. In one investigation, expression of B7-1 and B7-2 molecules was observed on murine astrocytes (7) while other groups have reported that murine and human astrocytes do not express B7 co-stimulatory molecules (11,12,28,30). Thus, in astrocytes, it is possible that there are both species and strain-related differences (8) in expression of B7 co-stimulatory molecules by astrocytes. The results presented in this report regarding B7 expression on astrocytes suggest an additional level of complexity. Here we show that primary and immortalized astrocytes from the same mouse strain differentially express B7-1 co-stimulatory molecules and that B7-1 expression correlates with their capability to serve as APC.

Previous studies have demonstrated phenotypic changes during astrocyte maturation (33,51) and that it is also possible to capture astrocytes at different stages of differentiation by immortalization (33). In this investigation we have clearly demonstrated that there are temporal differences in expression of B7-1 co-stimulatory molecules by astrocytes. Interestingly, in two of the three astrocyte lines immortalized on day 7, which do not express B7-1 proteins, we have detected B7-1 mRNA, suggesting that control of B7-1 protein expression in astrocytes may involve both transcriptional and post-transcriptional regulation (J. C. Patarroyo and S. S. Zamvil, unpublished observation). Differential expression of B7-1 and B7-2 protein molecules has also been observed in other cell lineages (52,53), including human peripheral blood dendritic cells that also exhibit differential B7 molecule expression based upon their stage of maturation (54,55). It is of interest that although astrocytes examined later could express B7-1 protein molecules, significant temporal differences in the expression level of other adhesion/co-stimulatory molecules, including ICAM-1, VCAM and CD40, were not observed.

B7-1 and B7-2 co-stimulatory molecules appear to have distinct roles in differentiation and amplification of Th1 and Th2 immune responses (18,19,22,25). In one study, although it was observed that BALB/c astrocytes could express both B7-1 and B7-2 co-stimulatory molecules, B7-2 provided B7:CD28 co-stimulation for both mature and naive CD4+ T cells specific for the foreign antigen, ovalbumin (7). In our investigation, we observed that IFN-{gamma}-stimulated B10.PL astrocytes examined both early and later up-regulated B7-2 co-stimulatory molecules, and that B7-2 provided B7:CD28 co-stimulation for the activation of mature encephalitogenic MBP Ac1–11-specific T cells. It is of interest that in a recent study of relapsing EAE, B7-2, but not B7-1, expression was observed in the CNS in the acute phase of EAE and that astrocytes in the active phases of EAE expressed B7-2 molecules (29). As activated encephalitogenic T cells can enter the CNS and initiate inflammation, it is possible that astrocytes that express B7-2 could contribute to the activation of these mature T cells. However, our results demonstrate that astrocytes which express B7-2, but not B7-1, do not stimulate Thp cells to differentiate into Th1 cells. Thus, it is unlikely that these astrocytes can participate in the recruitment of naive T cells involved in repertoire diversification that may occur in the later stages (15). It has also been suggested that astrocytes may serve to down-regulate CNS inflammatory processes through secretion of regulatory cytokines (13,14). In this regard, we have observed that although astrocytes that express B7-2, but not B7-1, do not stimulate proliferation of Thp, these astrocytes stimulate greater production of IL-4. It is therefore possible that these astrocytes (B7-1/B7-2+) could also participate in T cell regulation during recovery.

Certain studies suggest that B7-1 may promote Th1 inflammatory responses (18,19). In this study, discordant temporal B7 expression was observed, with B7-1 molecules appearing only on IFN-{gamma}-activated astrocytes examined later in culture. Not only were these astrocytes capable of activating mature encephalitogenic Th1 cells, they could provide a co-stimulatory signal for activation and Th1 differentiation of naive MBP-specific CD4+ T cells. It is also possible that this observation regarding activation of naive T cells by these astrocytes could reflect a requirement for a threshold level in B7 expression. It is of interest that B7-1 expression has been detected on astrocytes and infiltrating cells within the CNS in remission, between phases of active EAE (29). These investigators suggested that through its engagement with CTLA-4, B7-1 expression by these cells may contribute to the termination of proinflammatory responses, either by T cell inactivation or induction of apoptosis (29,56). Alternatively, based upon our findings regarding differential B7-1 expression and B7:CD28 co-stimulation, it is possible that B7-1 expression by astrocytes within the inflammatory lesions observed between clinical attacks of EAE may contribute to the recruitment of Th1 cells that could participate later in the course of EAE. It is also of interest that activated astrocytes in relapsing EAE have been observed to express CXC and CC chemokines that are chemoattractants for T cells, again suggesting that astrocytes may participate in the recruitment of T cells responsible for repertoire diversification in chronic CNS inflammation (5760). It is clear from both our study and Issazadeh et al. (29) that expression of B7-1 co-stimulatory molecules by astrocytes is not static. Although we did not observe temporal discordant expression of ICAM, VCAM and CD40, it is possible that in further analysis of these astrocytes we may discover temporal changes in the IFN-{gamma}-stimulated expression of other adhesion/co-stimulatory molecules that may have also contributed to differences in Thp activation and differentiation into Th1 and Th2 cytokine-producing cells that we have observed.

In vitro analyses, such as this one, serve as an important supplement for examining the potential function of individual cell populations in vivo (39,12). Results from this study, which demonstrate both temporal changes in B7-1 expression and differential activation of CD4+ T cells by separate astrocyte populations, suggest that astrocytes can have both proinflammatory and regulatory influences on CD4+ T cell activation. However, it is also recognized that propagation of astrocytes in vitro may introduce variables not encountered in vivo, which may influence their capability to regulate T cell activation. In further studies, examination of astrocytes isolated from the CNS at different stages of EAE and analysis of mice in which B7 expression has been targeted to astrocytes, may permit clarification of the role of B7 expression by astrocytes in vivo. Nevertheless, the results reported in this study underscore the importance for considering potential temporal changes in B7 expression when examining antigen presentation and T cell activation by astrocytes. Differential expression of B7-1 co-stimulatory molecules may be one mechanism involved in determining whether astrocytes participate in regulation or T cell activation in CNS inflammatory conditions in vivo.


    Acknowledgments
 
We thank Drs Vijay Kuchroo, Arlene Sharpe, Patricia Nelson, Anthony Slavin and Byron Waksman for insightful discussion and critical review of the manuscript. We are grateful to Drs J. Jacobberger and P. S. Jat for the SV40-406 cell line. J. M. S. is supported by a postdoctoral fellowship from the National Multiple Sclerosis Society. S. S. Z. is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society and is also supported by grant K08-NS01771 from the National Institutes of Health.


    Abbreviations
 
APCantigen-presenting cell
CVEcerebrovascular endothelial cell
EAEexperimental allergic encephalomyelitis
MBPmyelin basic protein
MSmultiple sclerosis
SEBstaphylococcal enterotoxin B

    Notes
 
Transmitting editor: L. Steinman

Received 18 December 1998, accepted 1 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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