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Central Nervous System APCs Inhibits T Cell Activation Capacity ofβIFN-

Ingrid Teige, Yawei Liu and Shohreh Issazadeh-Navikas

http://www.jimmunol.org/content/177/6/3542doi: 10.4049/jimmunol.177.6.3542

2006; 177:3542-3553; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/177/6/3542.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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IFN-� Inhibits T Cell Activation Capacity of Central NervousSystem APCs1

Ingrid Teige, Yawei Liu, and Shohreh Issazadeh-Navikas2

We have previously investigated the physiological effects of IFN-� on chronic CNS inflammation and shown that IFN-��/� micedevelop a more severe experimental autoimmune encephalomyelitis than their IFN-��/� littermates. This result was shown to beassociated with a higher activation state of the glial cells and a higher T cell cytokine production in the CNS. Because this statesuggested a down-regulatory effect of IFN-� on CNS-specific APCs, these results were investigated further. We report that IFN-�pretreatment of astrocytes and microglia (glial cells) indeed down-modulate their capacity to activate autoreactive Th1 cells. First,we investigated the intrinsic ability of glial cells as APCs and report that glial cells prevent autoreactive Th1 cells expansion whilemaintaining Ag-specific T cell effector functions. However, when the glial cells are treated with IFN-� before coculture with T cells,the effector functions of T cells are impaired as IFN-�, TNF-�, and NO productions are decreased. Induction of the T cellactivation marker, CD25 is also reduced. This suppression of T cell response is cell-cell dependent, but it is not dependent on adecrease in glial expression of MHC class II or costimulatory molecules. We propose that IFN-� might exert its beneficial effectsmainly by reducing the Ag-presenting capacity of CNS-specific APCs, which in turn inhibits the effector functions of encephali-togenic T cells. This affect is of importance because activation of encephalitogenic T cells within the CNS is a prerequisite for thedevelopment of a chronic progressive CNS inflammation. The Journal of Immunology, 2006, 177: 3542–3553.

T he type I IFN group member IFN-� is mainly producedby fibroblasts, macrophages, and dendritic cells in re-sponse to viral infection, dsRNA, nonvertebrate unmeth-

ylated DNA containing CpG motifs, or LPS (1–5). IFN-� signal-ing through the shared IFN-� or -� receptor triggers a cascade ofevents affecting both cells of the immune system as well as othercell types because the receptor is widely expressed (6). IFN-� isone of the therapeutic agents commonly used for treatment of mul-tiple sclerosis (MS),3 an inflammatory and demyelinating diseaseof the CNS (7, 8). There is a large amount of data available froma wide range of studies attempting to understand the basic mech-anisms of action for IFN-�. Among those, high doses of IFN-� inT cell cultures have been shown to induce a Th1 to Th2 shift andto reduce T cell proliferation (9–11). In accordance, several re-ports indicate that a Th1-Th2 shift occur in circulating T cells ofIFN-�-treated MS patients (12). It has also been reported thatIFN-� treatment causes professional APCs to produce less IL-12and more IL-10 that would favor Th2 responses over Th1 (13–15).Furthermore, it has been suggested that immune cells accessibility

to the CNS could be reduced upon IFN-� treatment by severalpossibilities. Among them is a down-regulation of several adhe-sion molecules important in leukocyte migration across the blood-brain barrier (e.g., LFA-1 and VLA-4) (16, 17). Next, stabilizingfunctions of endothelial tight junctions on cultured cells have beendemonstrated (18). In addition, the expression of matrix metallo-proteinases has been shown to be inhibited by IFN-� (19). How-ever, despite the efforts made to elucidate the subject, several re-ports concerning the effects of IFN-� on APCs (macrophages) andon T cells are contradictory, as both anti- and proinflammatoryproperties have been suggested. Regarding T cells, down-regulatedT cell response and a shift in Th1-Th2 response reported by somehave been challenged by others as it has been shown that IFN-�could induce a Th1 response (20–22). With reference to macro-phages, IFN-� has been shown to both increase and decrease se-cretion of TNF-�, IL-1�, and the soluble decoy receptor IL-1R�,depending on activation stimuli (23). Similarly, assaying PBMCsfrom treated MS patients show both up- and down-regulation ofcostimulatory molecules (24). This finding renders interpretationof data difficult and increases the demand for mimicking the invivo situation more closely for a better understanding of the basicmechanisms by which IFN-� exerts its beneficial effects. Many ofthe results obtained from MS patients have been achieved throughstudies of cells isolated from blood samples, although IFN-� couldhave profound effects on a number of different cell types consid-ering the broad expression of the IFN-� or -� receptor (6). It ispresumable that IFN-� could enter the CSF and hence access theCNS tissues due to leakage across blood brain barrier caused byincreased permeability during the course of disease. In addition,we have previously shown that IFN-� is indeed endogenously pro-duced within the CNS during experimental autoimmune enceph-alomyelitis (EAE) inflammation and could thus affect resident cellssuch as potential CNS APCs (25). So far, relatively little is knownregarding the effects of IFN-� on glial cells and their relative rolein regulation of autoreactive T cells at the site of inflammation.Only a handful of studies addressing this issue are available that

Neuroinflammation Unit, Section for Immunology, Institute for Experimental Medi-cal Science, Lund University, Lund, Sweden

Received for publication July 26, 2005. Accepted for publication June 20, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Swedish Foundation for StrategicResearch, Swedish Research Council-Natural Science, Swedish Research Council-Medicine, Alfred Osterlund Foundation, Tore Nilson Foundation, His Majesty GustavV Foundation, the Royal Swedish Academy of Science, Royal Physiographic Society,Lund, Sweden, the M. Bergvalls Foundation, Åke Wiberg Foundation, Borje DahlinFoundation, Segerfalk Foundation, and the Crafoord Foundation.2 Address correspondence and reprint requests to Dr. Shohreh Issazadeh-Navikas,Neuroinflammation Unit, Institute for Experimental Medical Science, BiomedicalCentre I13, Lund University, 221 84 Lund, Sweden. E-mail address: Shohreh.Issazadeh@med.lu.se3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental auto-immune encephalomyelitis; MBP, myelin basic protein; GFAP, glial fibrillary acidicprotein.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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report IFN-� inhibits glial cell expression of matrix metallopro-teinases 9 and 7 (26). Furthermore, IFN-� is able to reduce MHCclass II expression induced by IFN-� on microglial cells and re-sults in lower IL-12 production by microglia induced by LPS andIFN-�, in a similar manner to the APCs of lymphoid compartments(27).

In searching for a way to address the physiological mechanismsby which IFN-� could regulate immune response and the resultingCNS inflammation, we used IFN-� gene-deleted (IFN-� �/�) miceand EAE, the commonly used animal model for MS (25). Thestudy revealed an augmented and chronic EAE with increased in-cidence in IFN-��/� mice. This finding was associated with asignificantly elevated number of activated glial cells and higher invivo T cell cytokine production in the CNS of IFN-��/� micecompared with their wild-type (IFN-��/�) littermates. However,autoantigen-specific T cells isolated from peripheral lymphoid or-gans were similar in their production of cytokines and displayedequal encephalitogenic capacity as shown by adoptive transferstudies. These results suggested an increased activation of the en-cephalitogenic T cells in situ in IFN-��/� mice compared withIFN-��/�, resulting in an augmentation of the EAE. Hence, wehave now addressed the effects of IFN-� on the Ag-presentingcapacity of astroglia and microglia and report that treatment ofglial cells with IFN-� alone reduces their capacity to both induceand sustain an Ag-specific T cell cytokine production. Our dataindicate that one of the basic mechanisms by which IFN-� mightexert its beneficial effects could be due to its ability to down-modulate the Ag-presenting capacity of CNS glial cells, resultingin a less efficient reactivation of autoreactive T cells. Secondly,IFN-� suppresses the Ag-presenting capacity of the glial cells tomaintain effector functions of activated T cells.

Materials and MethodsMice

All mice used for cell cultures were of the B10.RIII strain. For EAE ex-periments, IFN-��/� mice generated as described previously (28) werebackcrossed for 12 generations to the B10.RIII strain, and these mice werecompared with their IFN-��/� littermates. All mice were bred and kept atthe conventional animal facility at Lund University (Lund, Sweden). Allexperiments were performed in accordance with the ethical committee inMalmo-Lund, Sweden.

Glial cell cultures

Primary mixed glial cell cultures were established from the forebrain andcerebellum of 1- to 2-day-old B10.RIII mice. The tissues were carefullydissected out and freed of meninges before being placed in HBSS solutionsupplemented with 1 mM pyruvate and 11 mM glucose. Tissue was thenchopped into smaller pieces using a razor blade and incubated with 1%trypsin (Sigma-Aldrich) for 10 min at 37°C. Thereafter, DNase and FCSwere added to a final concentration of 0.12 and 0.5%, respectively, andincubated for 8 min. Finally the solution was mechanically dissociated tosingle cell levels using a fire-constricted Pasteur pipette. The cells wereseeded at a concentration of 1.5 � 105 cells/cm2 in a 1:1 mix of DMEMand F12 medium (Invitrogen Life Technologies) supplemented with 0.16�M/ml penicillin, 0.03 �M/ml streptomycin, and 5% FCS. Medium waschanged every third to fourth day. After 7–10 days in culture, oligoden-drocytes were detached from the cell monolayer by shaking on an orbitalshaker. For expansion, the cells were split using a trypsin and EDTA so-lution (Sigma-Aldrich) upon reaching confluence. The cells were used forT cell coculture after two to three passages and consisted of a mixture of20–30% Mac-1� microglia and 70–80% glial fibrillary acidic protein(GFAP�) astrocytes. Pure microglial and astrocytic cultures were estab-lished as described (29). Briefly, after 8 days in culture, the mixed glialcells were vigorously shaken at 900-1000 rpm for 3 h on an orbital shaker.For microglial cultures, the floating cells were collected, washed, and re-seeded in 96-well plates (Nunc) at a concentration of 1 � 104 cells/well.After adhering overnight, nonadherent or loosely attached cells werewashed away. The adherent cells represented �97% pure microglial cellsas determined by Mac-1� staining, with �3% being GFAP�. For astrocyte

cultures, the still adherent cells were trypsinized and reseeded in flasks andleft to adhere for 30 min. Floating or loosely attached cells were recoveredby mild shaking by hand and the adhesion process was repeated, this timefor 1 h. Cells in the supernatant were thereafter collected, washed, andreseeded in 96-well plates (Nunc) at a concentration of 4 � 104 cells/well.These cells were �96% pure astrocytes as determined by GFAP� staining,�4% were Mac-1� cells. Both these cultures were used within 24–48 hafter establishment for coculture with T cells.

Establishment of myelin-specific T cell lines

Two T cell lines were generated by immunizing three 8- to 12-wk-old malemice in the hind paws and tail base with a total of 200 �l of a 1:1 emulsionof 250 �g of either myelin basic protein (MBP)89–101 or myelin oligoden-drocyte glycoprotein peptide 35–55 in PBS and CFA containing 100 �g ofMycobacterium tuberculosis H37Ra (Difco). Draining lymph nodes werecollected day 10 postimmunization and the cells went through severalrounds of Ag-specific restimulation as described (30). This generated T celllines of the Th1 phenotype that produced large amounts of IFN-� and IL-2and very low amounts of IL-4 upon Ag stimulation. In the experiments inour study, both MBP89–101-specific and myelin oligodendrocyte glycopro-tein peptide (35–55)-specific T cell lines have been used with very similarresults. Hence, no distinction will be made between them in the followingsections. In all experiments, the T cell lines had been subjected to from fourto eight stimulation rounds.

Coculture of T cells and glial cells

After the last trypsinization, glial cells were seeded in 96-well plates(Nunc) at 4–5 � 104 cells/well. After 24–48 h of adherence time, the cellswere either left untreated or treated for 24 h with 100 U/ml IFN-� orIFN-�. The cultures were then washed three times and T cells were addedin a 1:1 ratio. The T cell lines were established as previously discussed, andwere, in a first set of experiments, in a resting state (kept in medium sup-plemented with IL-2 for a minimum of 2 wk) when added to the differentlytreated glial cultures with or without the addition of Ag. These cells werereferred to as resting T cells. The concentration of the antigenic peptideswas 20 �g/ml if not stated otherwise. These cocultures were pulsed with[3H]thymidine after 48 h and harvested after 24 h of further incubation onglass fiber filters. [3H]Thymidine incorporation was measured in a betascintillation counter (Matrix 96 Direct Beta counter; Packard Instrument).Just before harvesting, supernatants were collected for cytokine analysis. Inparallel, some T cells were recollected after a total of 72 h in culture,stained for different activation markers, and analyzed in a four-color BDFACSort (BD Biosciences).

In a second set of experiments, the T cells were first activated for 48 hwith splenic APCs as described. They are referred to as activated T cells,which were washed thoroughly to remove Ag before being added to un-treated or IFN-�- or IFN-�-pretreated glial cells. These cultures were[3H]thymidine pulsed after 24 h in glial cocultures and harvested after 24 hof further incubation. As before, supernatants were collected and FACSstaining experiments were performed in parallel.

Transwell cocultures of T cells and glial cells

Mixed glial cultures were seeded at 2.5 � 105 cells/well in 24-well plates(Nunc). After 24–48 h of adherence, 2.5 � 105 T cells activated for 48 hwith splenic APCs (activated T cells) were added to the cultures in cellculture inserts (Transwells) with a pore size of 0.4 �m (BD Falcon). Thismethod allowed soluble molecules, but not cells, to pass between the gliasurface and the Transwell insert. As controls, glia and T cells plated at thesame density were cultured in 24-well plates without inserts, thus allowingcell-cell contact. After 24 h in coculture, the cells were [3H]thymidinepulsed and after an additional 24 h of incubation, the cells were harvestedand supernatants were collected.

Supernatant-mediated inhibition of T cell proliferation

Resting T cells were cocultured with glial cells in cell-cell contact cultureswith or without Ag (as described) and supernatants were collected understerile conditions. To investigate whether the supernatants could inhibit Tcell proliferation, resting T cells were activated with Ag and splenic APCsas described. This activation was performed in new medium or mediumconditioned with different ratios of the harvested supernatants. The prolif-eration index was calculated as proliferation in cultures with conditionedmedium divided by the control cultures containing 100% fresh medium.

FACS staining and evaluation

After 24 h of treatment with IFN-�, IFN-�, or a combination of these twocytokines (100 U/ml IFN-� for 12 h after which 100 U/ml IFN-� was

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added for the remaining 12 h), glial cell cultures were washed once withPBS and detached and dissociated using a trypsin and EDTA solution(Sigma-Aldrich). Activated or resting T cells were after 48 or 72 h incoculture, respectively, collected through vigorous flushing with the pi-pette. After washing in FACS buffer (2% FCS in PBS, for FACS of glialcells this buffer also contained 1 mM EDTA), cells were first incubatedwith anti-Fc receptor Ab (24.G.2, our hybridoma collection) at 10 �g/ml.Thereafter, glial cells were incubated with biotinylated FITC-marked orPE-marked Abs against Mac-1 (M1/70), MHC class II (Y3P or 7.16.17),CD1d (1B1), B7-1 (16-10A1), B7-2 (GL1), CD40 (3/23), ICAM-1 (3E2),VCAM-1 (429), Fas ligand (Kay-10), TGF-�1 (A75-3.1), and GFAP(Zymed Laboratories). For astrocytic GFAP staining and intracellularTGF-�1 staining, cells were first fixed using 3% paraformaldehyde andthereafter permeabilized using 0.3% saponin in FACS buffer. T cells werestained with biotinylated FITC- or PE-marked Ab against CD4 (Gk1.5),TCR �� (H57-597), CD28 (3751), CTLA-4 (UC10), CD40L (MR1),CD25 (7D4), CD69 (H1.2F3), and CD44 (Caltag Laboratories). Afterwashing, cells were incubated with streptavidin-PE or allophycocyanin di-luted 1/400 (BD Pharmingen). Annexin V and propidium iodide (BDPharmingen) staining was used to detect apoptotic and dead cells. In bothtypes of staining, all Abs were allowed to bind for 20 min on ice. All Abswere used at a concentration of 1–5 �g/ml and were purchased from BDPharmingen unless stated otherwise. The cells were analyzed using a four-color FACSort (BD Biosciences). Positivity was evaluated by comparisonof negative samples stained with irrelevant isotype-matched controls andstreptavidin-PE or allophycocyanin. Astrocytes were gated on GFAP, mi-croglia on Mac-1, and T cells on CD4 and TCR expression. For FACS-based proliferation studies, T cells were CFSE labeled at a concentration of5 nM before addition to glial cells. Cells were collected after 72 h andcounterstained with allophycocyanin-labeled anti-CD4 (Gk1.5). T cells la-beled just before FACS analysis were used as positive controls.

ELISA

Primary Abs: anti-IFN-� (5 �g/ml, clone R6A2; our hybridoma collec-tion), and anti-IL-2 and anti-TNF-� (2 and 6 �g/ml, clones JES6-1A12 andG281-2626, respectively; BD Pharmingen). Secondary biotinylated Abs:anti-IFN-� (0.6 �g/ml, clone Ani8; our hybridoma collection), and anti-IL-2 and anti-TNF-� (both used at 1 �g/ml, clones JES6-5H4 and MP6-XT3, respectively; BD Pharmingen). ELISA were performed as previouslydescribed (25)

NO assay

Nitrite was measured as the stable end product of NO and levels werequantified by a colorimetric assay based on the Griess reaction (31).

Induction and clinical evaluation of active EAE

MBP89–101 peptide (VHFFKNIVTPRTP-COOH; from Å. Engstrom, Uni-versity of Uppsala, Uppsala, Sweden) was used to induce EAE in IFN-��/� and IFN-��/� littermates. Mice were immunized s.c. at the base ofthe tail with 100 �l of emulsion of 250 �g of MBP89–101 peptide in PBSand CFA containing Mycobacterium tuberculosis H37Ra (Difco). In addi-tion, each animal received 400 ng of pertussis toxin (from Bordetella per-

tussis; Sigma-Aldrich) dissolved in 100 �l of PBS; this mix was given i.p.at the day of immunization and 2 days later.

Mice were sacrificed at day 13 postimmunization for immunohisto-chemistry during the acute phase of the EAE (25).

Immunohistochemistry

Brain and spinal cord were dissected out on day 13 postimmunization andimmediately embedded in OTC compound (Sakura Finetek) and snap-fro-zen in isopentane on dry ice. Tissues were cryosectioned in 10-�m slicesand kept at �70°C until staining. For single staining, the sections wereincubated with biotinylated anti-CD1d (1B1; BD Pharmingen) after whichExtrAvidin-peroxidase (Sigma-Aldrich) and diaminobenzidine (SaveenBiotech) were used for detection. Hematoxylin was used for backgroundstaining. For double staining, the tissues were again incubated with bio-tinylated anti-CD1d with streptavidin-Cy3 being used for detection. Tis-sues were simultaneously stained with FITC-labeled anti-NK1.1 (PK136;our own hybridoma collection).

Statistical analysis

When adding results from several experiments together, differences in pro-liferation, cytokine, and NO production were analyzed using a paired signt test. When comparing these parameters within single experiments, theStudent t test was used. Differences in FACS marker expression were an-alyzed with the Student paired t test. Values of p � 0.05 were consideredsignificant.

ResultsThe intrinsic capacity of glial cells as APCs is to preventfurther expansion of T cells while maintaining T cell effectorfunction

We were at first interested in determining whether there are dif-ferences between astrocytes and microglia in their Ag-presentingcapacity, as there has been a great deal of controversy in the lit-erature (32–34). As depicted in Fig. 1, there was no major quali-tative differences observed in between these two CNS-specificAPCs in their Ag-presenting capacity, measured by inducing T cellproliferation (Fig. 1A) and effector functions, determined by pro-duction of IFN-� (Fig. 1, B and C), TNF-�, and NO (data notshown). Furthermore, as shown in Fig. 1, B and C, there were nodiscrepancies observed between these two APC populations withregard to effect of cytokine treatment (IFN-� and IFN-�). Hence,we have used mixed culture of astrocytes and glial cells and re-ferred to them as glial cells. Interestingly, our analysis of the glialcells’ Ag-presenting capacity revealed that glial cells do not allowAg-specific T cell expansion, although they do induce Ag-specificT cells effector functions, measured by production of large amountof IL-2, IFN-�, TNF-�, and NO (Fig. 2). This result strongly in-dicates that the intrinsic function of CNS resident glial cells is to

FIGURE 1. Astrocytes and microglia fail to induce T cell proliferation while sustaining T cell IFN-� production. Glial cells cannot induce an Ag-specificT cell proliferation. A, CFSE-labeling of T cells in coculture with pure untreated astrocytes and Ag (solid black histogram) or with pure microglia and Ag(gray solid histogram) is shown. None of these cocultures show proliferating T cells. The hatched histogram shows the positive control of nondividing Tcells, that is, T cells CFSE labeled just before FACS analysis. Both astrocytes (B) and microglia (C) do induce an Ag-specific IFN-� response. Also, bothcell types respond similarly to IFN-� or -� treatment. The IFN-� response seen in microglial cultures without exogenously added Ag could be explainedby the presence of small amounts of endogenous myelin. No IFN-� was observed in any cultures of astrocytes or microglial cells only. Data show onerepresentative experiment each, and error bars represent SD of duplicate samples. �, p � 0.05; ��, p � 0.01.

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permit activated T cells to exert their effector functions once en-tering CNS, while still preventing them from further expansionin situ.

IFN-� negatively regulates the Ag-presenting capacity of glialcells by partly rescuing T cell expansion

It is commonly believed that development of a chronic, autoim-mune CNS inflammation is the result of activation of autoreactiveT cells in the peripheral lymphoid organs, which thereafter enterthe bloodstream and transverse the blood brain barrier. It is alsogenerally believed that these T cells require reactivation in situ toexert their deleterious effects (35, 36). Therefore, the function ofCNS resident APC populations (glial cells) could be detrimental inthe outcome of T cell reactivation in CNS. Thus, we investigatedthe effects of IFN-� on these cells and compared it with the knowneffect of a proinflammatory cytokine, IFN-�. We chose to studyboth cytokine effects on resting T cells, thus determining whetherglial cells could function as professional APCs, as well as on al-ready activated T cells. These T cells were activated using con-ventional irradiated splenic APCs and Ag (see Materials andMethods) for 48 h and thereafter washed and cocultured with glialcells. In this way, one could mimic interaction of activated T cellsentering the CNS with residual glial cells. Because there were nodifferences observed between specific T cell expansion usingCSFE labeling compared with DNA synthesis and thymidine up-take (see Figs. 1A and 2A), we use proliferation measured by thy-midine up-take as read out. Surprisingly, treatment of glial cellswith the proinflammatory cytokine IFN-� leads to significant Ag-specific inhibition of T cells proliferation (Fig. 3A). Similar find-ings were observed when activated T cells were used (Fig. 3B).Importantly, none of the differently treated glial cells induced sig-nificant T cell death as measured by annexin V and propidiumiodide staining (data not shown). Because these findings challengeseveral earlier reports regarding the APC role of glial cells in in-duction of T cell proliferation (32, 37, 38), we investigated whatcould be the reason for this discrepancy. As shown in Fig. 3C,irradiation of glial cells before coculture with resting T cells resultsin entirely different findings, as irradiated glial cells could induceT cell proliferation. Furthermore, IFN-� treatment does not haveany significant effect on T cell proliferation under this condition

(Fig. 3C). Very similar results were also achieved when usingmitomycin-treated glial cells (data not shown). Hence, irradiationand/or mitomycin exposure completely alters the APC function ofglial cells; it renders them capable of inducing Ag-specific prolif-eration. It might be assumed that this finding could partly be dueto the prevention of T cell expansion as a result of glial cell pro-liferation and crowding in nonirradiated cultures. However, thiswas addressed by using half the number of glial and T cells forcoculture and very similar results were achieved (data not shown).In addition, as shown earlier in Fig. 1A, CSFE labeling of T cellsbefore coculture with glial cells (which exclusively address T cellproliferation) verified the inability of glial cells to induce T cellproliferation.

IFN-� negatively regulates the Ag-presenting capacity of glialcells leading to inhibition of T cell effector function

As stated previously, our interest was to determine the effect ofIFN-�-treated glial cells on regulation and maintenance of T celleffector function. Thus, the capacity of resting and activated T cellsto produce IFN-� and TNF-� (two proinflammatory cytokines pro-duced by these encephalitogenic Th1 T cell lines) was measured inresponse to coculture with IFN-�- or IFN-�-treated glial cells. Nei-ther pure glial cells (astrocytes and microglia) nor mixed glialcultures produced any measurable IFN-� or TNF-� when culturedalone in the absence of T cells, regardless of exogenous cytokinestimulation (detection level 5–10 pg/ml) (data not shown). Thus,the measured IFN-� and TNF-� in the supernatant of coculturesmost likely represent the cytokine production by T cells. As de-picted in Fig. 4, A and C, IFN-� reduced Ag-specific IFN-� andTNF-� production by T cells. This result was in contrast to IFN-�,which as expected, enhanced the proinflammatory cytokine pro-duction of T cells. In cocultures using activated T cells, glial cellsinduced an enhanced T cell cytokine production, and this was to-tally prevented in IFN-�-treated cultures (Fig. 4, B and D). Be-cause it has been suggested that IFN-� can alter the phenotypicresponse by driving Th1 toward a Th2 phenotype, IL-4 was mea-sured in these cultures. However, we found no increase in IL-4production when the glial cells were pretreated with IFN-�, rathera tendency was observed toward a similar down-modulating effectresulting in lower amounts of IL-4 (data not shown). Hypotheti-cally, elevated levels of the immunosuppressive cytokine TGF-�1could possibly mediate the observed effects of IFN-� pretreatment.However, no TGF-�1 was detected in any supernatant, as mea-sured by ELISA (data not shown).

IFN-� treatment prevents glia-induced T cell up-regulation ofCD25

Aside from cytokine production, other markers generally used todefine T cell activation were also examined after coculture withIFN-� or -� treated or untreated glial cells. CD44 expression washigh in all cultures probably reflecting a memory phenotype of theencephalitogenic cell lines. CD69, in contrast, was expressed atrelatively low levels in all cultures (data not shown). It is possiblethat investigation of the cells at a different time point would revealdistinctions that were not observed in this study as CD69 is definedas an early activation marker (39). CTLA-4 expression was verylow on T cells from all cultures, whereas CD28 and CD40L werenot conclusively up- or down-regulated upon coculture with dif-ferently treated glial cells (data not shown). When investigating theactivation marker CD25 (the IL-2R �-chain), data well in accor-dance with the cytokine results were obtained. In cocultures ofresting T cells and IFN-� pretreated glial cells, a down-regulationof T cell CD25 expression was observed compared with T cellcocultures with untreated glial cells. IFN-� pretreatment increased

FIGURE 2. Glial cells inhibit T cell expansion but induce T cell effectorfunctions. A, Mixed glial cultures Ag-specifically inhibit T cell prolifera-tion but induce Ag-specific IL-2 (B), IFN-� (C), TNF-� (D), and NO pro-duction (E). Data represent three to nine individual experiments taken to-gether, and error bars represent SEM.

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the CD25 T cell expression. This observation was true for cultureswithout Ag addition as shown in Fig. 5A. In the presence of Ag, avery high CD25 up-regulation was observed in all cultures and theeffects of either IFN-� or IFN-� treatment were no longer observed(data not shown). Cocultures of activated T cells with glial cellsresulted in up-regulated T cell CD25 expression compared with Tcells cultured alone. These data again suggest that glial cells canperpetuate the T cell effector phase and thus support the findings ofcytokine production by T cells (Fig. 4, B and D). In accordance,IFN-� treatment of the glial cells totally inhibited the up-regula-

tion of CD25, while IFN-� treatment enhanced this up-regulationeven further (Fig. 5B). It is important to note that no IFN-� is leftin the cultures when the T cells are added, and IFN-� treatmentalone does not induce an autocrine loop of IFN-� production(which was measured by ELISA, data not shown). Hence, IFN-�is not acting on the T cells, but on the glial cells in the cultures.Taken together, these data suggest that IFN-� inhibits the capacityof potential CNS APCs to function as Ag-presenting cells activat-ing resting T cells. In addition, it prevents their capacity to sustainan ongoing T cell effector phase after Ag-specific activation.

FIGURE 3. Glia-mediated T cell proliferation arrestrequires living glia. A, Addition of Ag to cocultures ofnonirradiated glial cells and resting T cells gives a sig-nificantly suppressed T cell proliferation in IFN-�-treated cultures. IFN-� treatment results in higher pro-liferation compared with untreated glial cells. B, Theproliferation is also suppressed in cocultures of acti-vated T cells and IFN-�-treated glial cells (nonirradi-ated). C, Lethal irradiation of the glial cells just beforeT cell addition results in a totally different proliferationpattern. Untreated or IFN-�-treated glial cells then in-duce a high Ag-specific proliferation and no suppressionis seen in IFN-�-treated cultures. Data in A and B showthe mean � SEM of eight to nine experiments. Data inC show one representative experiment in which irradi-ated glial cells were used. Error bars represent SD oftriplicate samples. �, p � 0.05; ��, p � 0.01.

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The effects of glial cells on T cell responses is cell-cell contactdependent

Ag-specific activation of resting T cells is obviously dependent onMHC class II and TCR interaction, and therefore cell-cell contact.Nevertheless, we investigated whether the effects of glial cells onpreactivated T cells were mediated through a membrane-boundligand or through a soluble mediator, hence whether a Transwellsystem was used. As demonstrated in Fig. 6, A and B, both theeffects of untreated glial cells on preactivated T cell proliferationand cytokine production, as well as the relative effect of IFN-� or-� treatment, are inhibited once the cells are physically separatedby a membrane. This result strongly indicates that glial cells re-quire direct contact with T cells to exert their function. Still, thepossibility remained that this cell-cell interaction resulted in theproduction of a soluble mediator that could in return affect/inhibitT cell proliferation. To test this hypothesis, T cells were activated,this time with splenic APCs in the presence of different amounts ofconditioned medium from glial and T cell cocultures with or with-out Ag. The results obtained with supernatants from Ag-containingcultures are depicted in Fig. 6C and show that supernatant recov-ered from glia-T cell cocultures inhibit T cell proliferation in aconcentration-dependent manner. However, supernatants from un-treated, IFN-�- or IFN-�-treated cultures inhibited T cell prolifer-ation to the same extent. This outcome suggests that the differencesobserved between IFN-�- and IFN-�-treated glial cells were de-pendent on the expression of one or more cell surface moleculesyet undefined and not on soluble factors.

Glial cells inability to induce T cell proliferation is not due tohigh NO production or deficiency in IL-2 production

NO has previously been shown to inhibit T cell proliferation (40).Furthermore, it is well established that T cells require IL-2 forsuccessful proliferation (41) and it has been debated whether glialcells are capable of inducing an IL-2 response (33, 42–44). As

shown in Fig. 2B, the glial cells induced a clear Ag-specific IL-2response, and the T cell proliferation arrest could thus not be ex-plained by deficiencies in IL-2 production. To investigate whetherthe high NO production correlated with the observed inhibition ofT cell proliferation, the kinetics of these parameters were exam-ined. Fig. 7A demonstrates that proliferation increases over timeand after 96 h in the IFN-�-treated glial cell culture, the Ag-spe-cific inhibition of proliferation is in fact overcome. Next, Fig. 7Bshows that the NO production is also increasing over time andreaches a peak at 96 h, correlating with the highest level of pro-liferation. These data suggest that the inhibition of proliferation isnot due to high levels of NO production.

IFN-� treatment does not decreased MHC class II orcostimulatory expression on glial cells

The data presented in Fig. 6C suggested that differences in expres-sion of membrane-bound ligands could be responsible for the ob-served differences between T cell cocultures of untreated, IFN-�-treated, or IFN-�-treated glia. T cells generally require signalsthrough both MHC class II and costimulatory molecules for fullactivation and an effective Ag-specific response. FACS studieswere performed to investigate whether IFN-� treatment caused areduced expression of these molecules on glial cells, which couldexplain the achieved results. Surprisingly, no decrease was ob-served in any of the markers investigated in this model as result ofIFN-� treatment. Microglia and astrocytes were studied separatelyin these experiments based on Mac-1 or GFAP expression, respec-tively. MHC class II was not expressed in untreated astrocytes,IFN-� strongly up-regulated the molecule, and IFN-� alone had noeffect. In accordance with previously published data (45–47),IFN-� down-regulated an IFN-�-mediated MHC class II expres-sion on the astrocytes (Fig. 8A). The microglia population ex-pressed low levels of MHC class II and treatment with both IFNs

FIGURE 4. IFN-� treatment of glial cells reducesT cell production of IFN-� and TNF-�. T cells cocul-tured with IFN-�-treated glial cultures induce alower Ag-specific T cell IFN-� (A) and TNF-� (C)response compared with untreated controls. Pretreat-ment with IFN-� enhanced the cytokine response. Band D, When T cells preactivated with splenic APCsand Ag are added to glial cultures, untreated glialprolong the T cell cytokine production comparedwith T cells cultured alone. IFN-� pretreatment ofthe glial cells totally inhibits this enhancement andrestores the cytokine production to values indistin-guishable from T cells cultured alone. No IFN-� orTNF-� was observed in cultures of glial cells only.Data show the mean values of eight to nine individ-ual experiments, and error bars represent SEM ofthese experiments. �, p � 0.05; ��, p � 0.01.

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resulting in a MHC class II up-regulation (see Fig. 8A). The co-stimulatory molecules B7-1, B7-2, and CD40 were also studied.Astrocytes were shown to be highly positive for B7-1, whereasnegative for B7-2 and CD40 (see Fig. 8, B–D). None of thesemolecules was affected by IFN-� or IFN-� treatment alone, al-though the combination of both cytokines generated a tendencytoward up-regulation of B7-2 and CD40. In the microglia popula-tion, which was weakly positive for all these molecules when un-treated, both cytokines again generated higher expression of thesethree costimulatory molecules. An additive effect was observedwith respect to B7-2 expression, in which the combination of bothIFNs resulted in an even higher expression compared with anycytokine alone (see Fig. 8C). ICAM-1 and VCAM-1 were alsoinvestigated. ICAM-1 was shown to be relatively highly expressedin both astrocytes and microglia, but the expression was furtherup-regulated by both IFN-� and IFN-� on microglia (see Fig. 8E).The pattern was similar for microglial VCAM-1 expression (seeFig. 8F), whereas astrocytes expressed very high levels of VCAMthat were further up-regulated by the combination treatment withboth IFNs. Because glial cells have been shown to induce T cellapoptosis in a Fas ligand-dependent manner (48) and to secrete theimmunosuppressive cytokine TGF-�1 (49, 50), these moleculeswere also studied. However, no or very low expression was ob-served in glial cells and there was no marked effect as a result ofpretreatment with IFNs (data not shown). These results demon-strate that the suppressive effects that IFN-� treatment of glial cellsexerts on both T cell activation and effector phase maintenance is

not likely to be mediated through down-regulation of any of themolecules involved in classical T cell activation.

IFN-� and IFN-� elevate CD1d expression on glial cells

We also investigated the expression of the nonclassical MHC-likemolecule CD1d because we have reported that this molecule plays

FIGURE 5. IFN-� treatment of glial cells inhibits the up-regulation ofCD25 expression on T cells. A, IFN-� treatment of glial cells reduces theCD25 expression on subsequent cocultures with resting T cells in the ab-sence of Ag, whereas IFN-� treatment increases this compared with un-treated glial cells. Addition of Ag induces a very strong CD25 up-regula-tion on the T cells with no difference between treatments (data not shown).B, Coculture of activated T cells with untreated glial cells induces up-regulation of the T cell activation marker CD25 compared with T cellsculture alone. IFN-� pretreatment of the glial cells totally inhibits thisup-regulation, whereas IFN-� pretreatment increases the CD25 expressioneven further. Data show the mean value of three experiments. Fluorescencegeometric mean values � SEM are shown in parentheses. �, p � 0.05; ��,p � 0.01.

FIGURE 6. Glial cell-mediated perpetuation of activated T cell cyto-kine responses requires cell-cell contact. A, The IFN-�-mediated inhibitionof proliferation requires cell-cell contact as demonstrated by the fact thatinhibition is blocked in cultures where the T cells and glial cells are sep-arated by a membrane. Error bars represent SD of triplicate samples. B,When activated T cells and glial cells are allowed cell-cell contact, anincrease of the cytokine secretion by activated T cells is observed. Thisincrease is totally repressed in IFN-�-treated cultures and enhanced inIFN-�-treated cultures. When the T cells and glial cells are separated by amembrane, inhibiting direct cell-cell contact but allowing soluble mole-cules to pass freely, this cytokine production enhancement is blocked. Er-ror bars represent SD of triplicate samples. C, Different concentrations ofsupernatants from cocultures containing glial cells, T cells, and Ag wereadded to T cells stimulated by splenic APCs, and proliferation was mea-sured. Data show that there is a soluble mediator released upon cell-cellcontact in glia-T cell cocultures that can inhibit proliferation to a certainextent in a concentration-dependent manner. However, the differentlytreated supernatants have a similar inhibitory capacity. Error bars representSD of triplicate samples. ��, p � 0.01; ���, p � 0.001.

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a crucial role in the regulation of CNS inflammation (30). Inter-estingly, we found that both astrocytes and microglia express rel-atively high levels of CD1d and this was elevated by IFN-� as wellas by IFN-� (Fig. 9A). We continued to investigate how expressionof CD1d is regulated on glial cells in vivo and whether lack ofendogenous IFN-� influenced CD1d expression in the CNS. Asshown in Fig. 9B, CD1d (stained in red) is indeed up-regulated inglial cells in the inflamed CNS of EAE mice during the acute phaseof the disease. The CD1� cells are mainly astrocytes (Fig. 9B), asCD1 expression colocalized with GFAP expression (data notshown). Moreover, this reaction was associated with the recruit-ment of NK1.1� CD3� cells to the CNS. Fig. 9B shows NK1.1�

infiltrating cells (stained in green) in very close proximity to CD1-expressing astrocytes. Subsequent double staining with anti-CD3confirmed the presence of NK1.1� T cells (data not shown). How-ever, no difference was found between IFN-��/� mice and theirIFN-��/� littermates with respect to the regulation of CD1d ex-pression in glial cells during the course of EAE (data not shown).

DiscussionIt has recently been suggested that for the establishment of achronic inflammation in the CNS mediated by encephalitogenic Tcells (EAE), these T cells have to be efficiently reactivated in situ(51). This Ag-specific restimulation could be mediated by residualAPCs located in the CNS. We have previously reported, usingEAE model, that a deficiency in IFN-� leads to in situ activationof glial cells, the CNS-specific APCs, with a following activationof encephalitogenic Th1 cells, resulting in enhanced CNS inflam-mation and demyelination (25). This finding suggested that the

beneficial effects of IFN-� could be a result of negative modulationof the Ag-presenting capacity of glial cells. Thus, in this study weevaluate the effect of IFN-� on the capacity of the potential CNSAPCs (microglia and astrocytes) to induce and sustain activationof autoreactive Th1 cells. We also compared the effects of a proin-flammatory cytokine IFN-�.

Because the Ag-presenting capacity of glial cells has been con-troversial (37, 38, 52, 53) and differences have been suggestedbetween microglia cells and astrocytes in their APC functions (32–34), we first investigated these two glial cell types separately. Wefound no qualitative differences in Ag-presenting capacity of thesecell types, and no further differences were observed in their re-sponse to IFN-� or IFN-�. Interestingly, we found that both as-trocytes and microglia were capable of inhibiting expansion ofautoreactive Th1 cells, whereas, at the same time they sustain ef-fector functions of Th1 cells. Although this might seem contradic-tory at first glance, our findings are supported by previously re-ported in vivo data from EAE-affected animals, which suggeststhat autoreactive T cells infiltrating the CNS are indeed preventedfrom proliferating in situ (54, 55). Data presented by Ford et al.(56) are also supports our current findings. In their studies, adultmicroglia purified directly ex vivo induced insufficient T cell ac-tivation with inhibition of proliferation and IL-2 production de-spite ample production of TNF-� and IFN-� (40, 56). Similar re-ports on APC capacity of astrocytes have shown that they couldinduce cytokine production without a corresponding proliferation(57). However, our data suggest that interaction of glial cells withTh1 cells does not render them anergic because there is no defectin IL-2 production or up-regulation of CD25 (IL-2R �-chain). In

FIGURE 7. Proliferation arrest is not caused by high NO production. A, The kinetics of cell proliferation in glial cell (G) and T cell (T) cocultures isshown. Proliferation is expressed as a percentage of the proliferation in cultures of glial cells only. The inhibition of proliferation is Ag-dependent. Theinhibition gets less efficient over time and is finally overcome at 96 h in IFN-�-treated cultures. The kinetics of glial cell proliferation alone are not affectedby the treatments (data not shown). B, All glial cultures induce an Ag-specific NO production, which increases over time. The figure shows that the highestNO levels coincide with the highest T cell proliferation. Error bars represent SD of triplicate samples in all panels.

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fact, interaction of T cells with untreated glial cells alone is ade-quate to up-regulate CD25 and IL-2 production. The observed Ag-specific inhibition of T cell proliferation in the current study isdependent on cell-cell contact, which results in release of a solublesubstance that suppresses proliferation. The inhibition of T cellproliferation was only overcome in long-term (96 h) cocultureswith IFN-�-treated glial cells, which could be representative of apersistent inflammatory stimulus in vivo. In addition, when irra-diated or mitomycin-treated glial cells were used, a very strongAg-specific T cell proliferation was observed. Thus, the earlierconflicting in vitro results concerning the ability of glial cells toinduce or inhibit T cell proliferation (32, 37, 57, 58) could partly

be the result of the use of IFN-�-stimulated, mitomycin-treated, orirradiated and hence damaged glial cells in the majority of previ-ous reports (32, 37, 38, 52, 53). Prevention of proliferation re-ported in this study is not associated with NO production, whichwas suggested previously (40). Furthermore, we found no supportfor the involvement of TGF-�1 or Fas-Fas ligand interaction in theinhibition of T cell proliferation.

It is noteworthy to mention that only activated T cells pass thewell-protected CNS (59), hence these data indicate an intrinsicability of glial cells to permit activated T cells to exert their ef-fector function for which they have been predestined, while at thesame time inhibiting their expansion to protect the integrity of vital

FIGURE 8. IFN-� treatment of glial cells does not induce down-regulation of MHC class II or costimulatory molecules. Mixed glial cells consisted ofastrocytes and microglia distinguished by expression of GFAP and Mac-1, respectively. A, Astrocytes were negative for MHC class II when untreated orIFN-�-treated, but MHC class II was strongly up-regulated upon IFN-� stimulation. IFN-� reduced the IFN-�-mediated up-regulation. Untreated microgliaexpressed low amounts of MHC class II and this effect was further enhanced when treated with either IFN-� or IFN-�. B, Untreated astrocytes expressedhigh levels of B7-1, and the expression remained unchanged upon treatment. Microglia expressed medium levels of B7-1 and the expression increased upontreatment with IFN-� or IFN-�. C, Astrocytes were negative for B7-2 in all conditions. Untreated microglia expressed medium levels of B7-2, whereas theexpression was significantly up-regulated after treatment with IFN-�, IFN-�, or both in combination. D, Astrocytes were negative for CD40 in all culturesalthough a nonsignificant tendency toward CD40 up-regulation was seen in cultures treated with both IFN-� and IFN-�. Untreated microglia expressed lowlevels of CD40 and the expression was increased in cultures treated with IFN-� or IFN-�. E and F, Astrocytes express very high levels of ICAM andVCAM, respectively, which is further increased by combination treatment with both IFN-� and IFN-�. Untreated microglia expressed only low levels butup-regulated their expression upon treatment with either IFN-� or IFN-�.

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functions of CNS from being damaged by immune-mediated in-flammation. However, our data also indicate that persisting inflam-matory mediators might alter this function and bring about the glialcell capability of inducing T cell proliferation. This function couldin turn lead to prolonged activation of T cells with consequentchronic neuroinflammation as seen in chronic EAE and MS.

Importantly, in this study we also show that IFN-� decreases thecapacity of the glial cells to induce Ag-specific T cell effectorfunctions, as measured by IFN-�, NO, and TNF-� production.IFN-� thereby reduces the mediators that could lead to a chronicinflammation. This effect is also true for T cells that are already inan activated state when encountering the glial cells. Interestingly,untreated glial cells increased T cell cytokine production, as wellas induced CD25 up-regulation on preactivated T cells, whichspeaks for an inherent capacity to sustain T cell effector functionsindependent of Ag specificity. This activation was entirely blockedwhen glial cells were pretreated with IFN-� before interaction withT cells. The fact that similar results are obtained in both of theseconditions, of which the latter is devoid of Ag, strongly suggeststhat activation is mediated by a mechanism distinct from MHCclass II and TCR interaction. In theory, there is a possibility thattraces of Ag are still present in the culture medium loaded onto thesplenic APCs. However, when the CD25 expression was measured

on resting T cells, in which neither Ag nor APCs have been presentfor a minimum of 2 wk, CD25 was still up-regulated when T cellswere cocultured with untreated glial cells. This up-regulation wasprevented in IFN-�-treated glial cell cultures and this arguesagainst involvement of an Ag-dependent mechanism. Taken to-gether, our findings might shed light on the possible local effects ofIFN-� in other situations than direct IFN-� administration, wherethere is an endogenous up-regulation of IFN-� within the CNS,such as in the event of neuroinflammation (autoimmune or viralinduced) that could result in defective in situ immune regulation byglial cells with subsequent chronic neuroinflammation.

Other studies investigating how glial cells affect activated Tcells have taken another approach and included both APCs fromlymphoid organs and Ag in their glial-T cell cocultures (60, 61). Incontrast to our results, they found that astrocytes inhibit T celleffector responses as measured by IL-2 production, CD25 up-reg-ulation, and TCR down-regulation. Gimsa et al. (61) also sug-gested an astrocyte-mediated CTLA-4 up-regulation on T cells,which was not observed in the present study. The differences inexperimental setups could account for this controversy. Our glialand preactivated T cell cocultures are devoid of lymphoid APCsand Ag, and this setup would mimic a different milieu where theavailability of Ag and lymphoid APCs is limited.

The maintenance of T cell effector functions induced by glialcells could be mediated through costimulatory and adhesion mol-ecules constitutively expressed on glial cells. In accordance, boththe astrocytes and microglia displayed different constitutive ex-pression patterns of B7, CD40, ICAM-1, and VCAM-1. The po-tential capacity of astrocytes to act as APCs has been widely de-bated and much of it has centered on their B7 expression (37). TheB7-1 expression observed in our study is likely to be strain-specificbecause astrocytes from SJL mice have similarly been shown to bemainly expressing B7-1 (62, 63), whereas astrocytes from BALB/cmice are primarily expressing B7-2 (53).

On the contrary, the inhibitory effect of IFN-� is not likely to bemediated by signaling through the MHC class II-TCR pathway orcostimulatory signaling. None of the molecules we investigatedthat are involved in these pathways were down-regulated uponIFN-� treatment in either the astrocyte or microglia population.Rather the opposite was observed, as both MHC class II and sev-eral of the costimulatory molecules were up-regulated in the mi-croglia fraction. In agreement with our earlier reports (25, 64), noshift from Th1 to Th2 phenotype was detected as a result of IFN-�treatment of glial cells, as has been previously suggested by others(15, 65). These facts together strongly point toward a more gen-eralized down-modulatory effect of IFN-� on the stimulatory ca-pacity of CNS APCs. Interestingly, IFN-� was recently shown toup-regulate the inhibitory costimulatory molecule PD-L1 in den-dritic cells (66) and it remains to be investigated whether IFN-�has similar affect on glial cells. In one study (27), microglia wereactivated with IFN-� or LPS and the addition of IFN-� was shownto down-regulate T cell cytokine production. However, becauseIFN-� also reduced the IFN-�-induced MHC class II expression,this result alone could explain the reduction in T cell activation.Our report demonstrates the sole effects of IFN-� on glial APCfunctions.

Because our data suggested an immunoregulatory role of glialcells on encephalitogenic T cell expansion and function, it was ofparticular interest to investigate the regulation of CD1d on glialcells. CD1d is known to stimulate a subset of T cells with postu-lated immunoregulatory functions in EAE and MS (67–69), andwe have shown that mice deficient in CD1d develop augmentedand a more chronic EAE (30). Interestingly, we found that bothIFNs increased expression of CD1d on astrocytes and microglia.

FIGURE 9. Glial cells express CD1d NK1.1� infiltrating cells in closevicinity of CD1� glial cells in the CNS of EAE mice. A, Untreated astro-cytes and microglia expressed medium levels of CD1d but the expressionincreased upon treatment with either IFN-� or IFN-�. Data show the meanvalue � SEM for 10 experiments. Significant difference between untreatedand IFN-treated cultures is shown �, p � 0.05; ��; p � 0.01; and ���, p �0.001. B, NK1.1� infiltrating cells (green) in close interaction with CD1� glialcells (red). The image is taken around an infiltrate from the cerebellum of anEAE-affected B10.RIII wild-type mouse on day 13 after immunization.

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This observation is of value for future studies exploring CD1dregulation in the CNS, as this is the first report on cytokines ca-pable of up-regulating CD1d on glial cells. We also demonstratethat CD1d is up-regulated in vivo on glial cells of EAE-affectedmice. This finding is in accordance with previous reports in whichit has been shown that both MS patients (70) and EAE-affectedguinea pigs (71) display CD1 reactivity in the CNS. The majorityof the CD1d-restricted immunoregulatory T cells express the NKcell marker NK1.1. Indeed, we detected NK1.1� cells in closevicinity of CD1� astrocytes, indicative of a functional CD1-de-pendent Ag presentation by these glial cells. Nevertheless, despitethe up-regulatory effects of IFN-� on CD1d, there was no differ-ence in CNS CD1 expression between EAE-affected IFN-�-defi-cient mice and their wild-type littermates. Taking the in vitro andin vivo findings together, it could be concluded that IFN-� has aneffect on the induction of the CD1d molecule on glial cells, andthus could play a role in the regulation of CNS inflammationthrough this mechanism. However, this effect is not exclusivelyIFN-�-dependent and could be overcome by other factors up-reg-ulated in an inflammatory environment, such as IFN-�. EAE-af-fected IFN-�-deficient mice do have increased levels of IFN-� inthe CNS (25) and this could explain the maintained levels of CD1expression in these mice.

In summary, we report that the intrinsic role of glial cells is toinhibit expansion of T cells while allowing their effector function.Moreover, IFN-� suppresses the capacity of these potential CNSAPCs, both to induce an Ag-specific T cell cytokine response, andalso to sustain an ongoing T cell effector function. The effects ofIFN-� require cell-cell contact, but are not dependent on down-regulation of MHC class II or on any of the costimulatory or ad-hesion molecules involved in classical T cell activation. The cur-rent report might shed light on the basic mechanisms of IFN-�function during CNS inflammation and the possible partial bene-ficial effects of IFN-� on MS patients, as well as have implicationsfor other neuroinflammatory and neurodegenerative disease pro-cesses where glial activation is believed to be central to the diseaseprogression.

DisclosuresThe authors have no financial conflict of interest.

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