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the Absence of CD4 T CellsAntitumor CTL Effector Function, Even in The Brain Parenchyma Is Permissive for Full

Pierre-Yves DietrichNathalie Scamuffa, Philippe Saas, Nicolas de Tribolet and Paul R. Walker, Thomas Calzascia, Valérie Schnuriger,

http://www.jimmunol.org/content/165/6/3128doi: 10.4049/jimmunol.165.6.3128

2000; 165:3128-3135; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/6/3128.full#ref-list-1

, 27 of which you can access for free at: cites 61 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Brain Parenchyma Is Permissive for Full Antitumor CTLEffector Function, Even in the Absence of CD4 T Cells1

Paul R. Walker,2*† Thomas Calzascia,* Valerie Schnuriger,* Nathalie Scamuffa,*Philippe Saas,‡ Nicolas de Tribolet,† and Pierre-Yves Dietrich*

Effective antitumor immune responses against cerebral malignancies have been demonstrated in several models, but precisecellular function of specific effector cells is poorly understood. We have explored this topic by analyzing the MHC class I-restrictedT cell response elicited after implantation of HLA-CW3-transfected P815 mastocytoma cells (P815-CW3) in syngeneic mice. In thismodel, tumor-specific CTLs use a distinctive repertoire of TCRs that allows ex vivo assessment of the response by immunophe-notyping and TCR spectratyping. Thus, for the first time in a brain tumor model, we are able to directly visualize ex vivo CTLsspecific for a tumor-expressed Ag. Tumor-specific CTLs are detected in the CNS after intracerebral implantation of P815-CW3,together with other inflammatory cells. Moreover, despite observations in other models suggesting that CTLs infiltrating the brainmay be functionally compromised and highly dependent upon CD4 T cells, in this syngeneic P815-CW3 model, intracerebraltumors were efficiently rejected, whether or not CD4 T cells were present. This observation correlated with potent ex vivocytotoxicity of brain-infiltrating CTLs, specific for the immunodominant epitope CW3 170–179expressed on P815-CW3 tumorcells. The Journal of Immunology,2000, 165: 3128–3135.

T here is considerable current interest in immune responsesto tumors, as models for understanding various aspects ofimmune regulation and for the possibility of exploiting

such immune reactivity in clinical applications. Indeed, the everexpanding list of tumor Ags found in spontaneous human cancers(1, 2) now forces us to address the issue of why so many antigenictumors still escape immune control. For this we have recourse tomodel systems in which we have become increasingly adept atinducing or augmenting antitumor immunity using various vaccinestrategies, such that the host can then reject transplanted parentaltumors (3).

It was once thought that the immune privileged status of thebrain may pose an insurmountable challenge for antitumor im-mune responses. However, immune privilege is currently inter-preted as meaning an immune reactivity that is modified ratherthan absent (4). Indeed, lymphocyte entry to the brain (5, 6) and adegree of lymphatic drainage from the CNS are now well de-scribed (7). Furthermore, spontaneous immune responses havealso been documented in human glioma (8), although these areinsufficient to eliminate the tumor. More impressive antitumor ac-tivity has been demonstrated in different animal models, in whichresponses induced in the periphery are able to mediate antitumoreffects in the brain (9–15). Many of these approaches have beendeveloped from models for tumors in other sites, but there is not

always a direct translation of such therapies to brain tumors. Forexample, studies in which multiple cytokines have been tested asmodulators of immune responses gave different results accordingto the tumor model (SMA-560, B16) and site of implantation (13,16). Other attempts to create a cellular vaccine by overexpressionof ICAM-1 on a glioma cell line resulted in growth inhibition ofglioma cells implanted s.c., but not in the CNS (17). Furthermore,in certain clinical studies, it was suggested that tumors metasta-sizing to the CNS could be particularly resistant to treatment withadjuvant immunotherapy (18). In most of these studies, the im-mune (or indeed other) mechanisms responsible for the success orfailure of the immunomodulating therapy have been difficult todirectly address. In many cases, this is because certain rodent braintumor models are poorly characterized or are not always totallysyngeneic (12, 19, 20).

To facilitate immunological analyses, some recent studies haveused better characterized tumor models in the brains of syngeneicmice, such as B16/F10 melanoma (13, 21) and C3 sarcoma cellstransfected with the human papilloma virus type 16 (22). Thesemodels have provided useful information about ways of inducingefficient antitumor responses and of the different cellular and cy-tokine requirements for such immunity. Nevertheless, the specialrequirements for safe, efficient immune responses in the CNS ne-cessitate further information about the specificity of such re-sponses. However, there is very little in vivo data for the finespecificity of immune responses to brain tumors, principally be-cause of the difficulty to date in defining specific T cells. We havetherefore chosen to investigate immune responses to a brain tumorusing a model that facilitates identification of specific T cells. Thisis the immune response elicited in syngeneic DBA/2 mice afterimplantation of P815-CW3 tumor cells. It was demonstrated (innon-CNS sites to date) that P815-CW3 tumor cells are rejected bya potent CTL response focused on an immunodominant determi-nant from region 170–179 of the CW3 molecule (CW3170–179).These CTLs exhibit highly conserved TCR structural features, in-cluding the apparently exclusive usage of a BV10 TCR (23), andcan be readily monitored ex vivo by flow cytometry as an

*Laboratory of Tumor Immunology, Division of Oncology, and†Department of Neu-rosurgery, University Hospital Geneva, Geneva, Switzerland; and‡Laboratory of Im-munology, E.F.S. Bourgogne Franche Comte, Besancon, France

Received for publication February 23, 2000. Accepted for publication June 22, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Cancer Research Switzerland (to P.R.W., Grant KFS626-2-1998), the Fondation Ernst et Lucie Schmidheiny, the Fondation Kisane, theFondation Gustave-Prevot, and the Fondation pour la Lutte contre le Cancer et pourdes Recherches Biologiques.2 Address correspondence and reprint requests to Dr. Paul R. Walker, Division ofOncology, University Hospital Geneva, rue Micheli-du Crest 24, 1211 Geneva 14,Switzerland. E-mail address: Paul.Walker@hcuge.ch

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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expanded BV101CD62L2CD81 subset (24, 25) or by TCR spec-tratyping (26). The utility and accuracy of defining CW3-specificCTLs by either of these two techniques have also been confirmedby MHC/peptide tetramer analysis (26).

In this study, we exploit the unique characteristics of the im-mune response to P815-CW3 to address crucial issues about therecruitment and function of CTLs specific for a tumor located inthe CNS. We show that the majority of CD8 cells infiltrating thebrain parenchyma are CW3 specific and retain high functional ac-tivity even in the absence of CD4 cells.

Materials and MethodsCell lines

Transfection of the P815 murine mastocytoma with HLA-CW3 has beenpreviously described (27). Clone 444/C9.3.1 was used for all implanta-tions, hereafter referred to as P815-CW3, kindly supplied by Drs. J. L.Maryanski and J.-C. Cerottini (Ludwig Institute for Cancer Research, Lau-sanne, Switzerland). Cultured cells were washed and resuspended in PBSbefore implantations. For in vitro cytotoxicity tests, nontransfected P815cells were also used as targets.

Mice and P815 cell implantations

Adult female DBA/2 mice (RCC, Fullingsdorf, Switzerland, or IFFACredo, L’Arbresle, France) were implanted with viable P815-CW3 cellsfrom culture. For s.c. implantations, 23 107 cells were injected in theflank. For intracerebral (i.c.)3 implantation, mice were first anesthetized byinhalation of isoflurane (Abbott, Baar, Switzerland), followed by i.p. in-jection of a mixture of xylazine (Bayer, Leverkusen, Germany) and ket-amine (Warner-Lambert, Baar, Switzerland). P815-CW3 cells were thenimplanted into the pallidum with the aid of a syringe mounted in a stereo-taxic instrument (Stoelting, Indulab, Gams, Switzerland), 2.5 mm lateral tobregma and 3.5 mm below the surface of the skull. Preliminary dose-response experiments in which between 53 103 and 53 105 P815 cellswere implanted i.c. indicated that immune responses and clinical symptomswere more uniformly apparent when 53 105 cells were implanted, and sothis dose was used for all subsequent experiments. Higher numbers of cellscould not be implanted because the limit of weight loss permitted by thelocal animal welfare authorities had been reached.

Cell preparations

PBL were purified by Ficoll-Hypaque centrifugation (Pharmacia, Uppsala,Sweden), and single cell suspensions from lymph nodes and spleen wereprepared by standard procedures. For isolation of brain-infiltrating leuko-cytes (BILs), mice were sacrificed by CO2 asphyxia, then immediatelyperfused through the left cardiac ventricle with isotonic Ringer’s solution.Brains were removed and BILs were isolated by enzymatic digestion andmodified Ficoll-Hypaque centrifugation, as previously described (28). Theenzymes used in this procedure do not degrade the principal surface re-ceptors on T cells, allowing ex vivo functional and phenotypic analyses tobe performed without any preculture of cells.

Flow cytometry

The CW3-specific immune response was assessed by quantifyingBV101CD62L2CD81 cells (24). PBLs, splenocytes, lymph node cells, orBILs were triple stained with FITC-conjugated CD62L (Mel-14; Immuno-Kontakt, Frankfurt, Germany), PE-conjugated CD8 (53-6.7; PharMingen,San Diego, CA), and biotinylated anti-BV10b (B21.5; PharMingen) re-vealed with streptavidin-tricolor (Caltag Laboratories, Burlingame, CA).Additional analyses were performed using CD4 PE (KT6; Serotec, Oxford,U.K.); CD11b FITC (M1/70; PharMingen); CD4 biotin (RM4-4; Phar-Mingen), or CD11b biotin (M1/70, PharMingen), followed by streptavidin-tricolor (Caltag). Samples were analyzed using a FACScan equipped withCellQuest software (Becton Dickinson, Mountain View, CA). Wheremeans for pooled data are given, SEM are also indicated.

Immunohistology

Brains (perfused as described above) were snap frozen in 2-methylbutane(Merck, Wertheim/Main, Germany) and stored at280°C until used for

sectioning. Coronal sections (7mm) were made and fixed with 2% para-formaldehyde. After quenching of endogenous peroxidase, sections werestained with primary Abs. The following rat mAb were used as tissueculture supernatants prepared in our laboratory: anti-BV10 (B21.5) (29),CD4 (GK1.5, ATCC TIB207), CD8 (H35-17.2) (30), and antimacrophage/microglia (F4/80, ATCC HB 198). The secondary Ab was goat anti-rat Igcoupled to HRP (BioSource International, Camarillo, CA). Purified poly-clonal rabbit antisera were used to detect glial fibrillary acidic protein(Dako Diagnostics, Zug, Switzerland) and P1Ap (kindly supplied by Dr. A.Amar-Costesec) (31), followed by donkey anti-rabbit Ig coupled to HRP(Jackson ImmunoResearch, West Grove, PA). Peroxidase activity was re-vealed by adding a freshly prepared solution of AEC substrate (3-amino-9-ethyl-carbazole; Sigma, Buchs, Switzerland) and H2O2. Sections werecounterstained with hematoxylin and eosin.

In vivo depletion of CD4 T cells

In some experiments, mice were depleted of CD4 T cells by i.p. injectionof 0.2 mg of CD4 mAb (GK1.5, ATCC TIB 207) on days24, 22, and13,relative to P815-CW3 implantation. Depletion to at least 95% was con-firmed by flow cytometry of PBL the day before P815-CW3 implantationusing a noncompeting Ab (RM4-4, described above).

Cytolytic assay

P815 cells (or transfected P815 cells) were labeled with 150mCi of sodium[51Cr]chromate, as previously described (27) for 1 h at 37°C and washedthree times. For peptide pulsing, 10mM of peptide CW3170–179was addedduring the labeling procedure. A total of 120051Cr-labeled target cells wasmixed with varying numbers of freshly isolated effector cells in round-bottom microplates, the cells being resuspended in DMEM supplementedwith 5% FCS and HEPES.51Cr release in supernatants was measured after4 h of incubation at 37°C. The percent specific lysis was calculated as:100 3 ((experimental 2 spontaneous release)/(total2 spontaneousrelease)).

Size analysis of complementarity-determining region 3 (CDR3)of the TCR

The CDR3 region of the PCR-amplified TCR BV4 and BV10 transcriptswas analyzed using a run-off procedure, as previously described (8, 32, 33).Briefly, total RNA was prepared from perfused brain using TRIzol (LifeTechnologies, Paisley, U.K.) and converted to cDNA by standard methodsusing reverse transcriptase and an oligo(dT) primer. These cDNAs wereamplified using validated 59sense primers specific for either BV4 or BV10and one 39antisense primer specific for the BC gene segment (33). Ali-quots (2ml) of BV4-BC or BV10-BC PCR products were subjected to afive-cycle run-off reaction, using a dye-labeled oligonucleotide primer spe-cific for the BC segment. The run-off products were then run on an auto-mated sequencer in the presence of fluorescent size markers. The length ofthe DNA fragments and the fluorescence intensity of the bands were ana-lyzed with Immunoscope software (developed by C. Pannetier, PasteurInstitute, Paris, France).

ResultsMice implanted i.c. with P815-CW3 cells exhibit transientweight loss and a systemic antitumor immune response

Preliminary experiments in which different numbers of P815-CW3cells were implanted i.c. indicated that there were few externalclinical signs until 53 105 cells were used. At this cell dose, mostmice exhibited some transient weight loss and mild clinical symp-toms (ruffled fur, hunched posture). This cell dose was used for allsubsequent experiments. Neither mice injected i.c. with PBS, normice injected s.c. with P815-CW3 exhibited weight loss or othersymptoms.

Using flow cytometry, we screened blood and lymphoid tissueof mice implanted with P815-CW3 cells for evidence of CW3-specific CTLs (BV101CD62L2CD81 cells). As previously re-ported for i.p. immunized mice, i.c. implantation of P815-CW3also resulted in a significant expansion of CW3-specific CTLs, inboth spleen and PBL (Fig. 1A). Lymph nodes were also examined(axillary, inguinal, and cervical), but generally no significant pro-portions of CW3-specific CTLs were detectable (data not shown).

The site of implantation of P815-CW3 cells influenced the ki-netics of the systemic CW3-specific CTL response. Intracerebral

3 Abbreviations used in this paper: i.c., intracerebral; AEC, 3-amino-9-ethyl-carba-zole substrate; BIL, brain-infiltrating leukocyte; CDR, complementarity-determiningregion; DC, dendritic cell; d.p.i., days postimplantation.

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implantation resulted in a rapid specific CTL response in the blood(Fig. 1B) as well as the spleen (not shown). Indeed, as early as 7days postimplantation (d.p.i.), i.c. implanted mice showed an ele-vated proportion of CW3-specific CTLs in PBL that was signifi-cantly higher than in s.c. implanted mice (p 5 0.001, Mann-Whit-ney rank sum test). However, the response did not reach the samemagnitude and declined faster than the slower response inducedafter s.c. implantation. Intraperitoneal implantation shows similarkinetics and magnitude to s.c. implantation (data not shown andRef. 24), and i.v. injection results in either an undetectable (in mostmice) or a very low level response (data not shown).

Regression of i.c. P815-CW3 tumor correlates with leukocyteinfiltration of the brain parenchyma

Immunohistology of brain cryosections 5 days postimplantationrevealed significant tumor growth in the ipsilateral hemisphere rel-

ative to the site of tumor implantation; this could be readily visu-alized by the morphology of the P815-CW3 cells and by positivestaining with antiserum specific for the P815 tumor Ag P1Ap (Fig.2A). At this time point, there was also significant staining in theipsilateral hemisphere with F4/80 mAb, specific for macrophages,monocytes, and microglial cells (not shown). Strongest F4/80staining was in the peritumoral region, but some F4/801 cells alsoinfiltrated the tumor mass. Only rare CD4 or CD8 T cells weredetected at 5 d.p.i. (not shown).

By 8 d.p.i., few tumor cells remained (Fig. 2B), but there wassignificant infiltration of BV101 T cells (Fig. 2,C andD), CD8 Tcells (not shown), and CD4 T cells (Fig. 2,E andF). The presenceof F4/801 cells also persisted at day 8, with similar localization tothat found for infiltrating lymphocytes (not shown). The vast ma-jority of infiltrating T cells was found in the ipsilateral hemisphere,in perivascular locations as well as infiltrating the brain paren-chyma. In some sections, the site of tumor implantation could belocalized and this region was usually well infiltrated (sometimesintensely) with leukocytes.

Brain sections were also examined at later time points, at 11, 14,17, 21, and 22 d.p.i.; tumor cells could not be detected at these timepoints, but the inflammatory infiltrate persisted, albeit at lowerlevels. The other notable difference to the brains taken 8 d.p.i. wasthat infiltrating cells were widely distributed throughout both ce-rebral hemispheres; this is illustrated for BV101 T cells (Fig. 2,GandH), and comparable results were found for cells positive forCD4, CD8, and F4/80 (at time points from day 11 onward). Cellswere found in most regions of the brain: intraparenchymal,perivascular, periventricular, and sometimes in the lateralventricles.

Dominant oligoclonal expansions of BV101 T cells expressing a6-aa CDR3 region are detected in brains of mice implanted i.c.with P815-CW3

Two of the principal features of the T cell repertoire specific forthe H-2Kd-restricted immunodominant epitope CW3170–179 ex-pressed by P815-CW3 cells are the BV10 selection and the usageof a 6-aa CDR3 region (23, 34). The flow cytometry and immu-nohistochemistry data indicated that BV101 cells had been ex-panded; the CDR3 size was examined by TCR spectratyping (Fig.3). Isolation of RNA was conducted on brains that had been wellperfused to eliminate leukocytes present in vessels. RT-PCR wasperformed using primers specific either for BV10 or BV4 TCRgene segments, the latter having no known preferential usage in theresponse to P815-CW3. The curves obtained for BV4 and BV10for spleen from control mice (Fig. 3) showed a bell-shaped form,consistent with polyclonal populations of T cells, with most CDR3sizes represented. However, for brains of mice previously im-planted i.c. with P815-CW3, curves obtained for BV10 (Fig. 3)showed a single predominant peak corresponding to 6 aa, consis-tent with an expanded population of BV101 T cells expressing anappropriate TCR for recognition of CW3170–179, whereas the pro-file for BV4 was similar to that found for control mice.

Most CD8 T cells isolated from brains of mice implanted i.c.with P815-CW3 express a BV10 TCR and are functionallycytotoxic ex vivo

BILs were isolated from mice implanted i.c. 8 days previouslywith P815-CW3, a time point at which immunohistochemistry dataindicated that there were high numbers of leukocytes infiltratingthe brain parenchyma and at which a high level systemic immuneresponse (.50% BV101 cells in the CD62L2CD81 subset) wasdetectable in the PBL of most (36/47) mice tested. An average of

FIGURE 1. Mice implanted i.c. with P815-CW3 mount a systemic im-mune response to P815-CW3. Peripheral blood was taken before and 11days after i.c. injection of 53 105 viable P815-CW3 cells. At this time,mice were sacrificed and spleen cells were isolated. Cells were triplestained with CD62L, CD8, and anti-BV10 mAb and analyzed by flowcytometry.A, CD8 cells were gated for low (a) or high (b) expression ofCD62L and analyzed for BV10 expression, as shown in the correspondinghistograms.B, Proportions of P815-CW3-specific cells (percentage ofBV101CD62L2CD81 cells in the lymphocyte gate) were calculated fromflow cytometric analysis at various times after i.c. implantation of 53 105

viable P815-CW3 cells (n5 33) or PBS (n5 14) or after s.c. injection with2 3 107 P815-CW3 cells (n5 19). Curves represent pooled data (6SEM);not all mice were analyzed at all time points.

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106 leukocytes was isolated from the brain of each mouse; isola-tions from control mice that had not been implanted i.c. yielded toofew cells to count accurately, but probably no more than 53 104

cells per brain. The enzymes (collagenase D and DNase I, togetherwith the trypsin inhibitorNa-p-tosyl-L-lysine chloromethyl ke-tone) and method of dissociation had no effect on expression levelsof CD4, CD8, BV10 TCR, CD62L, or CD11b (as judged by pre-liminary experiments with control cells), nor did enzyme treatmentaffect cytotoxic function of a control CTL clone. Therefore, thephenotype and function of isolated BILs were tested immediatelyafter isolation, allowing conclusions to be made regarding thelikely in vivo characteristics of these cells.

Among mice showing a high level systemic response to CW3,flow cytometric analysis showed that the immune infiltrate con-sisted of mainly CD8 cells, CD4 cells, and CD11b1 cells (mac-rophages or microglial cells). Only very low numbers of B cells,NK cells, and granulocytes were found in preliminary experi-ments, so in view of the limited number of cells available foranalysis, these populations were not routinely stained. Consistentwith the notion that only activated T cells can enter the CNS (35),more than 95% of both CD4 and CD8 cells were negative forCD62L expression. Analysis of BILs triple stained with CD8,CD62L, and anti-BV10 TCR Abs (Fig. 4A) showed that a highproportion of the CD62L2CD81 cells used a BV10 TCR (gener-

ally more than 70%), whereas BV10 usage by CD4 cells was al-ways less than 10%. This result (also consistent with the immu-nohistochemistry and TCR molecular analysis) encouraged us totest the specific cytotoxic function of BILs ex vivo against P815cells pulsed with a synthetic peptide corresponding to the immu-nodominant CTL epitope CW3170–179(Fig. 4B). Freshly isolatedBILs were highly and specifically cytotoxic, killing P815 cellsonly when the specific peptide was present. Furthermore, whenE:T ratios were calculated based on the numbers ofBV101CD62L2CD81 cells (thus encompassing the vast majorityof cells defined in many previous studies as being able to recognizeCW3170–179), this high cytotoxic activity was manifested at evenmodest E:T ratios. These results are therefore consistent with theBV101 CD81 T cells infiltrating the brain parenchyma (detectedin vivo by various techniques) as being fully differentiated CTLs.

The anti-P815-CW3 CTL response elicited after i.c. implantationis CD4 independent during both the induction and effectorphase

It is not possible to generalize about the CD4 dependence of CTLresponses in the brain, because very few studies have looked at thisaspect in systems enabling the analysis of specific CTL responses.We therefore investigated this issue in the response to P815-CW3,depleting mice of CD4 T cells before tumor cell implantation and

FIGURE 2. Immunohistochemical detectionof tumor and lymphocytes infiltrating brains ofmice implanted with P815-CW3 cells. Coronalcryosections were stained with polyclonal ormonoclonal primary Ab, followed by HRP-con-jugated second Ab, and revealed by AEC. Orig-inal magnification was340 for all photomicro-graphs.Top row, P815-CW3 tumor cells in theipsilateral hemisphere stained with anti-P1Apantiserum at 5 d.p.i. (A) or 8 d.p.i. (B). The re-maining pairs of photomicrographs show ipsilat-eral hemispheres on theleft-hand sideand con-tralateral hemispheres on theright-hand side.Cand D, BV101 cells stained with B21.5 mAb,8 d.p.i.;E andF, CD4 cells stained with GK1.5mAb, 8 d.p.i.; G and H, BV101 cells stainedwith B21.5 mAb, 14 d.p.i.

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maintaining the depleted state throughout the response. Intraperi-toneal injection of CD4 mAb (GK1.5) resulted in CD4 T cell de-pletion to more than 95% of normal CD4 T cell numbers (in PBL),as tested by flow cytometry the day before i.c. implantation ofP815-CW3. A further i.p. injection of depleting CD4 mAb wasadministered 3 d.p.i., and at sacrifice virtually no CD4 T cells weredetected in the brain parenchyma, by flow cytometry or immuno-histochemistry (data not shown). However, the absence of CD4 Tcells did not inhibit the infiltration of BV101 cells into the brainparenchyma, as observed by immunohistochemical staining of day8 brain cryosections (Fig. 5A). Furthermore, BILs could be isolatedfrom brains of depleted mice, exhibiting comparable phenotypeand function as for nondepleted mice (Fig. 5,B andC). The ma-jority of mice tested (9/13, 69%) showed a comparable infiltrationof CTLs to nondepleted mice.

DiscussionIn this study, we have examined the function of CTLs specific foran Ag expressed by P815-CW3 tumor cells growing in the CNS.This model does not aim to recreate all of the multiple and com-plex interactions occurring between spontaneous brain tumors andtheir host (something never really achieved by any transplantabletumor model), but it does have some unique advantages. It enablesthe ex vivo identification of CD8 cells specific for an immuno-dominant epitope expressed by P815-CW3, whether or not thesecells are fully functional effector cells. The choice of a system thatfacilitates the analysis of CD8 CTLs is important because we con-sider that this cell type may be a key effector cell potentially ca-pable of potent antitumor immunity. CD8 cells are certainly not theonly candidates and they may be highly dependent upon other celltypes for their activation, expansion, migration, differentiation, andfunction. This study is a first step in a defined system to exploresome of these issues in the context of an Ag expressed on tumorcells located in the brain.

The most basic question we address in this study is that of im-mune function in the CNS. Although P815-CW3 cells efficiently

induce CW3-specific CTLs in other sites (24, 25, 36), it was un-known whether this would be the case in the CNS. Indeed, themany reports of immunosuppression associated with brain tumors(37, 38) and even with normal brain microenvironment (19, 39, 40)made this question highly relevant. The short-term growth of i.c.tumors followed by their regression (Fig. 2), accompanied by asystemic specific immune response (Fig. 1) and minor clinicalsymptoms, suggested that tumor-specific immunity was occurring.What was surprising was the rapidity of the specific response com-pared with that achieved after s.c. implantation (Fig. 1B) or afteri.p. implantation (24), as measured by flow cytometry of PBL andsplenocytes. However, these data only reflect the tissue that is

FIGURE 3. Expanded populations of T cells using a BV10 TCR with a6-aa CDR3 region are detected in the brains of mice implanted i.c. withP815-CW3. The CDR3 size distribution profiles of PCR-amplified TCRBV4-BC and BV10-BC transcripts were analyzed, as described inMate-rials and Methods. The patterns obtained show the size and intensity dis-tribution of in-frame BV-BC amplification products; the size of the CDR3region is deduced from the fragment length. Relative fluorescence intensityis plotted on the vertical axis, with graphs normalized to 100% for the mostintense peak. The profiles for spleen are from control DBA/2 mice; thosefor brain are from mice implanted i.c. 21 days previously with P815-CW3.

FIGURE 4. The majority of T cells isolated from brains of mice im-planted with P815-CW3 are CW3-specific CTLs exhibiting specific cyto-toxicity ex vivo. Mice were sacrificed 8 days after i.c. implantation of 53105 viable P815-CW3 cells. After perfusion, brains were removed andBILs were isolated, as described inMaterials and Methods.A, Cells weretriple stained with anti-BV10, CD62L, and CD8 mAb and analyzed by flowcytometry. CD8 cells (32.1% of BILs) were gated for low expression ofCD62L (a) and analyzed for BV10 expression, as shown in the correspond-ing histogram. Staining profiles representative of 15 of 17 analyses, ofBILs from either individuals or pools of mice.B, Freshly isolated cellswere tested for cytotoxic function in a 4-h51Cr release assay, using P815cells (E) or P815 cells pulsed with peptide CW3170–179(F) as targets. TheE:T ratio was calculated either using the total number of lymphocytesadded to the wells (All Cells), or the number of BV101CD62L2CD81

cells, estimated from the flow cytometric data. Similar levels of specificcytotoxicity were obtained in nine independent assays.

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tested and may not detect expanded populations of cells at thetumor site or in draining lymphoid tissue. Nevertheless, for i.c.implanted mice, a strong leukocyte infiltration of the brain paren-chyma was detected by immunohistochemistry at about 8 d.p.i.(Fig. 2). We sequentially refined the identification of the infiltrat-ing cells by 1) staining with mAb to BV10 (Fig. 2); 2) TCR mo-lecular analysis of perfused brain tissue that revealed expandedpopulations of BV101 T cells with a 6-aa CDR3 region (Fig. 3);3) flow cytometric analysis of leukocytes isolated from perfusedbrain, indicating large numbers of BV101CD62L2CD81 T cells(Fig. 4A); and finally 4) ex vivo functional analysis of leukocytesisolated from perfused brain that efficiently killed P815 cellspulsed with peptide CW3170–179or P815-CW3 (not shown), butnot unmodified P815 cells without peptide (Fig. 4B). Taken to-gether, these data convincingly indicate that large numbers ofCW3-specific CTLs were induced after i.c. implantation of P815-CW3 and that they infiltrated the brain and were specificallycytotoxic.

Analysis of the immunohistochemistry data gives further insightinto the likely sequence of events leading to tumor elimination.The tumor is clearly visible at day 5 after implantation, but at thisstage there are very few T cells infiltrating the brain. Moreover, atthis stage in blood or spleen, there is no detectable expansion ofBV101CD62L2CD81 T cells by flow cytometry or of BV101

cells with a 6-aa CDR3 by TCR spectratyping (data not shown).However, there is evidence for innate immune reactions, becausemany F4/801 cells are present. Cells of this phenotype can corre-spond to resident parenchymal microglial cells as well as perivas-cular and recruited peripheral macrophages (41, 42). The presenceof these microglia/macrophages is probably a critical step for lym-phocyte infiltration, because in macrophage-depleted mice, acti-vated leukocytes can extravasate from blood across CNS endothe-lium, but cannot penetrate the parenchyma. Instead, theyaccumulate in the perivascular and subarachnoid spaces (43).However, microglia/macrophage activation and recruitment maynot be the exclusive mechanism responsible for the developmentof the early immune response, because astrocytes may also play arole (44–46). Indeed, glial fibrillary acidic protein expression byastrocytes was high in the ipsilateral hemisphere at 5 d.p.i. (datanot shown), one of the signs of reactive gliosis that accompaniesmany forms of CNS stress (47). Astrocytes in such an activatedstate can participate in immune reactions by up-regulation of MHCand adhesion molecules (44, 48, 49) and liberation of inflamma-tory chemokines and cytokines (45, 46, 50, 51). Analysis of eventseven nearer to the day of implantation is difficult to interpret in atransplanted tumor model because of the inevitable physicaltrauma associated with implantation, but these issues of the initi-ating events for an antibrain tumor response are evidently impor-tant to address in future studies.

The significant lymphocyte accumulation at the tumor site by8 d.p.i. also corresponds to the peak of weight loss and the ap-pearance of mild clinical symptoms in some mice. Because by thistime the tumor burden is reduced compared with day 5, the im-mune response rather than the tumor may be responsible for thesymptoms. Very few lymphocytes are present in the normal brain(or indeed in the nontumor-laden contralateral hemisphere of theimplanted mice investigated in this study; Fig. 2), but activated

FIGURE 5. The induction and effector phases of the specific CTL re-sponse to the immunodominant epitope expressed by P815-CW3 cells im-planted i.c. can occur in the absence of CD4 T cells. Mice were depletedof CD4 T cells by two i.p. injections of GK1.5 CD4 Ab, then implanted i.c.with of 5 3 105 viable P815-CW3 cells. One further injection of mAb wasperformed, then mice were sacrificed and perfused 8 d.p.i. Brains wereremoved and used either for isolation of BILs or for immunohistochemis-try. A, Coronal cryosections were stained with mAb to BV10, followed byan HRP-conjugated second Ab, and revealed by AEC. The photomicro-graph shows BV101 cells infiltrating the ipsilateral hemisphere, originalmagnification340. B, Isolated BILs were triple stained with anti-BV10,CD62L, and CD8 mAb and analyzed by flow cytometry. CD8 cells (69.2%of BILs) were gated for low expression of CD62L (a) and analyzed forBV10 expression, as shown in the corresponding histogram. Staining pro-files representative of 9 of 13 analyses.C, Freshly isolated cells were testedfor cytotoxic function in a 4-h51Cr release assay, using P815 cells (E)

or P815 cells pulsed with peptide CW3170–179(F) as targets. The E:T ratiowas calculated either using the total number of lymphocytes added to thewells (All Cells), or the number of BV101CD62L2CD81 cells, as assessedby flow cytometry. Similar levels of specific cytotoxicity were obtained infour independent assays.

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lymphocytes can traverse the blood brain barrier (35), for example,as may occur after initial T cell priming at a peripheral site. How-ever, whether Ag or intact tumor cells (19) reach secondary lym-phoid organs is far from clear and is not an issue that has beendirectly addressed in this study. Once an inflammatory response isinitiated in the CNS, naive T cells may also contribute to the im-mune infiltrate (52).

The initially restricted localization of infiltrating T cells is con-sistent with retention at the site of highest Ag concentration. TheMHC class I-restricted CTLs can presumably interact directly withH-2Kd-expressing P815 cells, whereas the abundant CD4 cellsmay recognize Ags expressed by Ia1 microglial cells or macro-phages, although the specificity of the CD4 cell component of thisresponse has not been characterized. It was also suggested thatthere is not only recruitment and retention of specific T cells, butalso the possibility of local expansion (53). The more widespreaddistribution of the immune infiltrate at 11 d.p.i. or later, after mostof the tumor cells have been eliminated (as judged by immuno-histochemistry), may be a consequence of the lack of retention ofcells in the ipsilateral hemisphere due to the less frequent encoun-ters with Ag. Studies of viral immune responses have indicated therelatively long-term persistence of CD8 T cells in the CNS, evenwhen only noninfectious viral Ags remain (54), although theseCTLs had lost their cytolytic capacities. In the model we describein this work, it is possible that once P815-CW3 cells have beeneliminated, tumor Ag might persist on APC in the brain even 3 wkafter implantation. However, by this stage there were too fewCTLs for us to isolate to examine their function.

Depletion of CD4 T cells appeared to have little effect on theinfiltration and function of CW3-specific CTLs (Fig. 5). This as-pect of the P815-CW3 response has previously been investigatedin other sites, with the response dependent upon CD4 cells whenP815 cells were injected i.p., but CD4 independent when they wereinjected intradermally (25). It was suggested that the abundantepidermal Langerhans cells and dermal dendritic cells (DC) in theskin (compared with the less efficient peritoneal macrophages pre-dominant in the peritoneum) were able to take up Ag from P815-CW3 and prime specific CTLs even in the absence of CD4 cells;however, such cross-priming has not yet been directly demon-strated for P815-CW3. As far as the CNS is concerned, we canonly speculate whether a local professional APC can capture Agand migrate to lymphoid tissue in the absence of CD4 activation(or whether intact P815 cells exit the brain, as discussed previous-ly). Classical DC are not usually found in the brain, although his-tological reports have noted their presence in the choroid plexus(55, 56). Perhaps a more probable candidate for capturing CNS Agis the perivascular cell, which can take up Ag via scavenger re-ceptor-mediated endocytosis and subsequently migrate towardlymphoid tissue (recently discussed in Ref. 57), although any CD4dependence of these mechanisms has not been reported.

Because we depleted CD4 cells before tumor implantation andmaintained the depleted state throughout the tumor rejection phase,CTL effector function in the brain parenchyma was also indepen-dent of CD4 cells. This is not unprecedented, because in one in-teresting recent report, the CTL response against B16 melanomaimplanted i.c. was also CD4 independent, although only the effec-tor stage of this response was studied (13). Moreover, a CD4 in-dependence at both induction and effector stages of a CD8 T cellantitumor response was also found in an immunization modelagainst an i.c. sarcoma, but in this system, endogenous APC mayhave been bypassed because the vaccine consisted of cultured,Ag-pulsed DC (22). However, antiviral CTL responses in the CNSare often strictly CD4 dependent, and CTL function and/or viabil-ity have been reported to be lost in the absence of CD4 T cells (58,

59). Maintenance of CTL function in the brain is clearly dependentupon many factors that differ according to the model used. In theP511 mastocytoma model, CD8 T cells were unable to differentiateinto effector cells in the brain microenvironment (19), subse-quently attributed to a sensitivity to TGF-b present in the cere-brospinal fluid and the interstitial fluid (40). However, most of theexperiments were performed in outbred or nonsyngeneic mice,making it difficult to compare with the syngeneic response to adefined peptide Ag that we describe in this work.

In view of the advances in our understanding of antitumor im-mune responses, it is now tempting to contemplate the develop-ment of immunotherapies for spontaneous cerebral malignanciesin humans, particularly for tumors such as glioblastoma, for whichno adequate treatment exists. Typically, these approaches can beexpected to modify both specific and nonspecific components ofthe immune response. This has particular significance for the CNS,which has probably acquired mechanisms to limit inflammation,essential in a site that has severe physical limitations within theconfines of the skull to accommodate tissue swelling. Uncontrolledinflammation can lead to severe neurological dysfunction and maycontribute to the initiation or perpetuation of autoimmune disease.Indeed, early tumor immunology studies noted that lethal allergicencephalomyelitis was induced in different species by immuniza-tion with human glioma tissue (60). Furthermore, very recent ev-idence in a rodent gene therapy model for glioblastoma suggestedthat i.c. immune responses may directly or indirectly perpetuatereactive gliosis and demyelination (61). Taken together, such ob-servations argue for a thorough understanding of not only the cel-lular subsets required for antitumor effects, but also for the spec-ificity of these responses. The data from the P815-CW3 model thatwe describe in this work suggest that a highly focused responsecan mediate potent specific cytotoxicity against tumor cells in thebrain, even in the absence of CD4 T cells. Moreover, this antitu-mor activity occurred without untoward clinical symptoms. Thismodel opens the possibility to explore the factors that precludesuch an efficacious response against spontaneous glioblastoma,and how they may be overcome in future immunotherapies.

AcknowledgmentsWe thank A. Amar-Costesec, J.-C. Cerottini, A. Hugin, S. Izui, and J. L.Maryanski for generously providing cell lines or Abs, and P. Francois forinvaluable help in establishing the procedures for stereotaxic implantation.

References1. Boon, T., P. G. Coulie, and B. Van den Eynde. 1997. Tumor antigens recognized

by T cells.Immunol. Today 18:267.2. Wang, R. F., and S. A. Rosenberg. 1999. Human tumor antigens for cancer

vaccine development.Immunol. Rev. 170:85.3. Pardoll, D. M. 1995. Paracrine cytokine adjuvants in cancer immunotherapy.

Annu. Rev. Immunol. 13:399.4. Fabry, Z., C. S. Raine, and M. N. Hart. 1994. Nervous tissue as an immune

compartment: the dialect of the immune response in the CNS.Immunol. Today15:218.

5. Hickey, W. F., B. L. Hsu, and H. Kimura. 1991. T-lymphocyte entry into thecentral nervous system.J. Neurosci. Res. 28:254.

6. Irani, D. N., and D. E. Griffin. 1996. Regulation of lymphocyte homing into thebrain during viral encephalitis at various stages of infection.J. Immunol. 156:3850.

7. Cserr, H. F., and P. M. Knopf. 1992. Cervical lymphatics, the blood-brain barrierand the immunoreactivity of the brain: a new view.Immunol. Today 13:507.

8. Perrin, G., V. Schnuriger, A.-L. Quiquerez, P. Saas, C. Pannetier, N. de Tribolet,J. M. Tiercy, J. P. Aubry, P.-Y. Dietrich, and P. R. Walker. 1999. Astrocytomainfiltrating lymphocytes include major T cell clonal expansions confined to theCD8 subset.Int. Immunol. 11:1337.

9. Aoki, T., K. Tashiro, S. Miyatake, T. Kinashi, T. Nakano, Y. Oda, H. Kikuchi,and T. Honjo. 1992. Expression of murine interleukin 7 in a murine glioma cellline results in reduced tumorigenicity in vivo.Proc. Natl. Acad. Sci. USA 89:3850.

10. Asai, A., Y. Miyagi, H. Hashimoto, S. H. Lee, K. Mishima, A. Sugiyama,H. Tanaka, T. Mochizuki, T. Yasuda, and Y. Kuchino. 1994. Modulation oftumor immunogenicity of rat glioma cells by s-mycexpression: eradication of ratgliomas in vivo.Cell Growth Differ. 5:1153.

3134 CTL FUNCTION AGAINST A CNS TUMOR

by guest on May 11, 2018

http://ww

w.jim

munol.org/

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nloaded from

11. Resnicoff, M., C. Sell, M. Rubini, D. Coppola, D. Ambrose, R. Baserga, andR. Rubin. 1994. Rat glioblastoma cells expressing an antisense RNA to the in-sulin-like growth factor-1 (IGF-1) receptor are nontumorigenic and induce re-gression of wild-type tumors.Cancer Res. 54:2218.

12. Trojan, J., T. R. Johnson, S. D. Rudin, J. Ilan, and M. L. Tykocinski. 1993.Treatment and prevention of rat glioblastoma by immunogenic C6 cells express-ing antisense insulin-like growth factor I RNA.Science 259:94.

13. Sampson, J. H., G. E. Archer, D. M. Ashley, H. E. Fuchs, L. P. Hale, G. Dranoff,and D. D. Bigner. 1996. Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD81 cell-mediated immunity against tumorslocated in the “immunologically privileged” central nervous system.Proc. Natl.Acad. Sci. USA 93:10399.

14. Kikuchi, T., T. Joki, Y. Akasaki, T. Abe, and T. Ohno. 1999. Antitumor activityof interleukin 12 against interleukin 2-transduced mouse glioma cells.CancerLett. 135:47.

15. Graf, M. R., M. R. Jadus, J. C. Hiserodt, H. T. Wepsic, and G. A. Granger. 1999.Development of systemic immunity to glioblastoma multiforme using tumor cellsgenetically engineered to express the membrane-associated isoform of macro-phage colony-stimulating factor.J. Immunol. 163:5544.

16. Sampson, J. H., D. M. Ashley, G. E. Archer, H. E. Fuchs, G. Dranoff, L. P. Hale,and D. D. Bigner. 1997. Characterization of a spontaneous murine astrocytomaand abrogation of its tumorigenicity by cytokine secretion.Neurosurgery 41:1365.

17. Kikuchi, T., T. Joki, Y. Akasaki, T. Abe, and T. Ohno. 1999. Induction of an-titumor immunity using intercellular adhesion molecule 1 (ICAM-1) transfectionin mouse glioma cells.Cancer Lett. 142:201.

18. Mitchell, M. S. 1989. Relapse in the central nervous system in melanoma patientssuccessfully treated with biomodulators.J. Clin. Oncol. 7:1701.

19. Gordon, L. B., S. C. Nolan, H. F. Cserr, P. M. Knopf, and C. J. Harling-Berg.1997. Growth of P511 mastocytoma cells in BALB/c mouse brain elicits CTLresponse without tumor elimination.J. Immunol. 159:2399.

20. Beutler, A. S., M. S. Banck, A. Aguzzi, D. Wedekind, and H. J. Hedrich. 1997.Curing rat glioblastoma: immunotherapy or graft rejection?Science 276:20.

21. Ashley, D. M., B. Faiola, S. Nair, L. P. Hale, D. D. Bigner, and E. Gilboa. 1997.Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNAinduce antitumor immunity against central nervous system tumors.J. Exp. Med.186:1177.

22. Okada, H., H. Tahara, M. R. Shurin, J. Attanucci, K. M. Giezeman-Smits,W. K. Fellows, M. T. Lotze, W. H. Chambers, and M. E. Bozik. 1998. Bonemarrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effec-tive anti-tumor immunity against intracranial neoplasms.Int. J. Cancer 78:196.

23. Casanova, J.-L., J. C. Cerottini, M. Matthes, A. Necker, H. Gournier, C. Barra,C. Widmann, H. R. MacDonald, F. Lemonnier, B. Malissen, et al. 1992. H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors oflimited diversity.J. Exp. Med. 176:439.

24. Walker, P. R., T. Ohteki, J. A. Lopez, H. R. MacDonald, and J. L. Maryanski.1995. Distinct phenotypes of antigen-selected CD8 T cells emerge at differentstages of an in vivo immune response.J. Immunol. 155:3443.

25. Bour, H., C. Horvath, C. Lurquin, J. C. Cerottini, and H. R. MacDonald. 1998.Differential requirement for CD4 help in the development of an antigen-specificCD81 T cell response depending on the route of immunization.J. Immunol.160:5522.

26. Bousso, P., A. Casrouge, J. D. Altman, M. Haury, J. Kanellopoulos,J. P. Abastado, and P. Kourilsky. 1998. Individual variations in the murine T cellresponse to a specific peptide reflect variability in naive repertoires.Immunity9:169.

27. Maryanski, J. L., R. S. Accolla, and B. Jordan. 1986. H2-restricted recognition ofcloned HLA class I gene products expressed in mouse cells.J. Immunol. 136:4340.

28. Irani, D. N., and D. E. Griffin. 1991. Isolation of brain parenchymal lymphocytesfor flow cytometric analysis: application to acute viral encephalitis.J. Immunol.Methods 139:223.

29. Necker, A., N. Rebai, M. Matthes, E. Jouvin-Marche, P. A. Cazenave,P. Swarnworawong, E. Palmer, H. R. MacDonald, and B. Malissen. 1991. Mono-clonal antibodies raised against engineered soluble mouse T cell receptors andspecific for Va8-, Vb2- or Vb10-bearing T cells.Eur. J. Immunol. 21:3035.

30. Pierres, M., C. Goridis, and P. Golstein. 1982. Inhibition of murine T cell-me-diated cytolysis and T cell proliferation by a rat monoclonal antibody immuno-precipitating two lymphoid cell surface polypeptides of 94,000 and 180,000 mo-lecular weight.Eur. J. Immunol. 12:60.

31. Amar-Costesec, A., D. Godelaine, B. Van den Eynde, and H. Beaufay. 1994.Identification and characterization of the tumor-specific P1A gene product.Biol.Cell 81:195.

32. Pannetier, C., J. Even, and P. Kourilsky. 1995. T-cell repertoire diversity andclonal expansions in normal and clinical samples.Immunol. Today 16:176.

33. Pannetier, C., M. Cochet, S. Darche, A. Casrouge, M. Zoller, and P. Kourilsky.1993. The sizes of the CDR3 hypervariable regions of the murine T-cell receptorb chains vary as a function of the recombined germ-line segments.Proc. Natl.Acad. Sci. USA 90:4319.

34. Maryanski, J. L., C. V. Jongeneel, P. Bucher, J. L. Casanova, and P. R. Walker.1996. Single-cell PCR analysis of TCR repertoires selected by antigen in vivo: ahigh magnitude CD8 response is comprised of very few clones.Immunity 4:47.

35. Wekerle, H., C. Linington, H. Lassmann, and R. Meyermann. 1986. Cellularimmune reactivity within the CNS.Trends Neurosci. 9:271.

36. MacDonald, H. R., J.-L. Casanova, J. L. Maryanski, and J.-C. Cerottini. 1993.Oligoclonal expansion of major histocompatibility complex class I-restricted cy-tolytic T lymphocytes during a primary immune response in vivo: direct moni-toring by flow cytometry and polymerase chain reaction.J. Exp. Med. 177:1487.

37. Roszman, T., L. Elliott, and W. Brooks. 1991. Modulation of T-cell function bygliomas.Immunol. Today 12:370.

38. Walker, P. R., P. Saas, and P.-Y. Dietrich. 1997. The role of Fas ligand (CD95L)in immune escape: the tumor cell strikes back.J. Immunol. 158:4521.

39. Irani, D. N. 1998. The susceptibility of mice to immune-mediated neurologicdisease correlates with the degree to which their lymphocytes resist the effects ofbrain-derived gangliosides.J. Immunol. 161:2746.

40. Gordon, L. B., S. C. Nolan, B. R. Ksander, P. M. Knopf, and C. J. Harling-Berg.1998. Normal cerebrospinal fluid suppresses the in vitro development of cyto-toxic T cells: role of the brain microenvironment in CNS immune regulation.J. Neuroimmunol. 88:77.

41. Frei, K., C. Siepl, P. Groscurth, S. Bodmer, and A. Fontana. 1988. Immunobi-ology of microglial cells.Ann. NY Acad. Sci. 540:218.

42. Hickey, W. F., and H. Kimura. 1988. Perivascular microglial cells of the CNS arebone marrow-derived and present antigen in vivo.Science 239:290.

43. Tran, E. H., K. Hoekstra, N. Van Rooijen, C. D. Dijkstra, and T. Owens. 1998.Immune invasion of the central nervous system parenchyma and experimentalallergic encephalomyelitis, but not leukocyte extravasation from blood, are pre-vented in macrophage-depleted mice.J. Immunol. 161:3767.

44. Shrikant, P., and E. N. Benveniste. 1996. The central nervous system as an im-munocompetent organ: role of glial cells in antigen presentation.J. Immunol.157:1819.

45. Aloisi, F., A. Care, G. Borsellino, P. Gallo, S. Rosa, A. Bassani, A. Cabibbo,U. Testa, G. Levi, and C. Peschle. 1992. Production of hemolymphopoietic cy-tokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes inresponse to IL-1b and tumor necrosis factor-a. J. Immunol. 149:2358.

46. Saas, P., J. Boucraut, A.-L. Quiquerez, V. Schnuriger, G. Perrin, S. Desplat-Jego,D. Bernard, P. R. Walker, and P.-Y. Dietrich. 1999. CD95 (Fas/Apo-1) as areceptor governing astrocyte apoptotic or inflammatory responses: a key role inbrain inflammation?J. Immunol. 162:2326.

47. Eddleston, M., and L. Mucke. 1993. Molecular profile of reactive astrocytes:implications for their role in neurologic disease.Neuroscience 54:15.

48. Aloisi, F., G. Borsellino, P. Samoggia, U. Testa, C. Chelucci, G. Russo,C. Peschle, and G. Levi. 1992. Astrocyte cultures from human embryonic brain:characterization and modulation of surface molecules by inflammatory cytokines.J. Neurosci. Res. 32:494.

49. Rosenman, S. J., P. Shrikant, L. Dubb, E. N. Benveniste, and R. M. Ransohoff.1995. Cytokine-induced expression of vascular cell adhesion molecule-1(VCAM-1) by astrocytes and astrocytoma cell lines.J. Immunol. 154:1888.

50. Aloisi, F., G. Borsellino, A. Care, U. Testa, P. Gallo, G. Russo, C. Peschle, andG. Levi. 1995. Cytokine regulation of astrocyte function: in-vitro studies usingcells from the human brain.Int. J. Dev. Neurosci. 13:265.

51. Barnes, D. A., M. Huston, R. Holmes, E. N. Benveniste, V. W. Yong, P. Scholz,and H. D. Perez. 1996. Induction of RANTES expression by astrocytes and as-trocytoma cell lines.J. Neuroimmunol. 71:207.

52. Krakowski, A. M., and T. Owens. 2000. Naive T lymphocytes traffic to inflamedcentral nervous system, but require antigen recognition for activation.Eur. J. Im-munol. 30:1002.

53. Musette, P., J. F. Bureau, G. Gachelin, P. Kourilsky, and M. Brahic. 1995. Tlymphocyte repertoire in Theiler’s virus encephalomyelitis: the nonspecific in-filtration of the central nervous system of infected SJL/J mice is associated witha selective local T cell expansion.Eur. J. Immunol. 25:1589.

54. Bergmann, C. C., J. D. Altman, D. Hinton, and S. A. Stohlman. 1999. Invertedimmunodominance and impaired cytolytic function of CD81 T cells during viralpersistence in the central nervous system.J. Immunol. 163:3379.

55. Serot, J. M., B. Foliguet, M. C. Bene, and G. C. Faure. 1997. Ultrastructural andimmunohistological evidence for dendritic-like cells within human choroidplexus epithelium.NeuroReport 8:1995.

56. Hanly, A., and C. K. Petito. 1998. HLA-DR-positive dendritic cells of the normalhuman choroid plexus: a potential reservoir of HIV in the central nervous system.Hum. Pathol. 29:88.

57. Hickey, W. F. 1999. Leukocyte traffic in the central nervous system: the partic-ipants and their roles.Semin. Immunol. 11:125.

58. Stohlman, S. A., C. C. Bergmann, M. T. Lin, D. J. Cua, and D. R. Hinton. 1998.CTL effector function within the central nervous system requires CD41 T cells.J. Immunol. 160:2896.

59. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh,J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence ofactivated T cells without effector function.J. Exp. Med. 188:2205.

60. Bigner, D. D., O. M. Pitts, and C. J. Wikstrand. 1981. Induction of lethal exper-imental allergic encephalomyelitis in nonhuman primates and guinea pigs withhuman glioblastoma multiforme tissue.J. Neurosurg. 55:32.

61. Dewey, R. A., G. Morrissey, C. M. Cowsill, D. Stone, F. Bolognani, N. J. Dodd,T. D. Southgate, D. Klatzmann, H. Lassmann, M. G. Castro, et al. 1999. Chronicbrain inflammation and persistent herpes simplex virus 1 thymidine kinase ex-pression in survivors of syngeneic glioma treated by adenovirus-mediated genetherapy: implications for clinical trials.Nat. Med. 5:1256.

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