JOURNAL OF VIROLOGY, Apr. 1989, p. 1756-17620022-538Xl891041756-07$02.00/0Copyright C) 1989, American Society for Microbiology

HLA Class 11-Restricted Presentation of Cytoplasmic Measles VirusAntigens to Cytotoxic T Cells

STEVEN JACOBSON,' RAFICK P. SEKALY,2t CONNIE L. JACOBSON,'HENRY F. McFARLAND,' AND ERIC 0. LONG2*

Neuroimmunology Branch, National Institute of Neurological and Communicative Disorders and Stroke,' andLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases,

Building 4, Room 213,2 Bethesda, Maryland 20892

Received 14 July 1988/Accepted 22 December 1988

To analyze the nature of the HLA class II-restricted cytotoxic T-lymphocyte (CTL) response to measles virus,murine fibroblasts were transfected with expressible cDNA clones for human HLA-DR antigen and for measlesvirus matrix or nucleocapsid proteins. DR-positive murine fibroblasts transfected with measles virus matrix or

nucleocapsid genes were lysed by class II-restricted measles virus-specific CTL lines. Lysis was as efficient aswith infected autologous B-cell lines, even though the measles virus cytoplasmic proteins were undetectable byantibodies in the transfected target cells. These results demonstrate that cytoplasmic viral antigens can bepresented to CTL in the context of HLA class II antigens and that measles virus matrix and nucleocapsidproteins contribute to class II-restricted measles virus-specific CTL' responses. These results also show thatendogenously synthesized measles virus proteins can be efficiently presented by class II antigens. Theimplications of these findings for measles virus pathogenesis and for antigen processing are discussed.

Cytotoxic T lymphocytes (CTL) specific for virus-infectedcells recognize viral antigens in the context of self majorhistocompatibility complex (MHC) antigens expressed at thesurface of the target cells (44). Virus-specific CTL can beeither MHC class I-restricted CD8+ cells or MHC classII-restricted CD4+ cells. The frequencies of class I- andclass II-restricted CTL after in vivo infection vary betweenviruses. For instance, influenza virus elicits both class I- andclass II-restricted CTL, but the CTL response to measlesvirus is primarily class II restricted (16, 27; for reviews, seereferences 5 and 15).The particular antigens involved in class I-restricted CTL

recognition have been identified for several viruses. With thebest-studied example, influenza virus, the bulk of classI-restricted CTL elicited during in vivo infection in bothmice and humans recognizes determinants carried by theinternal nucleoprotein antigen (24, 36, 43). Influenza virus-specific class I-restricted CTL also recognize determinantson other internal antigens, such as matrix and polymerase,and only a minor subpopulation is directed towards the cellsurface glycoprotein hemagglutinin (for a review, see refer-ence 39). Class I-restricted CTL specific for several otherviruses are also directed predominantly to internal antigens(for a review, see reference 34).The requirement for processing of cytoplasmic viral anti-

gen for presentation to class I-restricted CTL was firstestablished by Townsend et al. (37). It is still unknown howcytoplasmic antigens are processed and how they cross themembrane that separates them from the peptide-binding siteon MHC molecules. The cell surface antigen hemagglutininis also processed for presentation by class I MHC molecules(4). Interestingly, exogenously added hemagglutinin did notsensitize target cells for class I-restricted CTL recognition,but endogenously synthesized hemagglutinin did (27). Newsynthesis is not a requirement for target cell sensitization as

* Corresponding author.t Present address: Clinical Research Institute of Montreal, 111

Pine West Avenue, Montreal, Quebec H2W 1R7, Canada.

shown by fusion with heat-inactivated influenza virus (42).Thus, the main requirement for class I-restricted CTL rec-ognition seems to be the presence of antigen in the cyto-plasm of target cells.

In contrast, little is known about viral antigens involved inclass II-restricted CTL recognition. Class II-restricted CTLresponses have been reported for influenza virus (10, 27),Epstein-Barr virus (26), herpes simplex virus (30, 40), andmeasles virus (16). Specificities for the cell surface hemag-glutinin and for the cytoplasmic nucleoprotein antigens weredetermined for different influenza virus-specific class II-restricted clones (10, 27). Two hemagglutinin-specificclones, in contrast to class I-restricted CTL, recognized onlyexogenously added, and not endogenously synthesized,hemagglutinin (27). Thus, the processing requirements forhemagglutinin presentation to class II-restricted CTL appearto be the same as for presentation of soluble antigens tohelper T cells. It is not yet known how cytoplasmic antigensare processed for presentation by MHC class II moleculesand whether they contribute significantly to class II-re-stricted CTL responses. Because CTL responses to certainviruses are primarily class II restricted (16, 30), it is impor-tant to define viral antigens that can be presented by MHCclass II molecules.To test whether cytoplasmic measles virus antigens con-

tribute to the CTL response, antigen presentation to measlesvirus-specific class Il-restricted CTL has been analyzed.Cells transfected with expressible cDNAs encoding humanMHC class II alpha and beta chains and with cDNAsencoding measles virus matrix (MV-M) or nucleocapsid(MV-N) proteins were efficiently lysed by specific CTLlines. Because the viral antigens originate only from endog-enous synthesis in the transfected cells, these results raiseinteresting questions about processing pathways for classII-restricted antigen presentation.

MATERIALS AND METHODSPlasmids. RSV.3 was constructed by adding the BamHI

(filled with Klenow polymerase)-HindIII linker fragment for

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HLA-DR-RESTRICTED PRESENTATION OF CYTOPLASMIC ANTIGENS 1757

RSVLTR

MV- cDNASV40

EBVoriP

RNA

FIG. 1. RSV.3 cDNA expression vector. The RSV.3 plasmidwas constructed as described in Materials and Methods. The pro-

moter sequence from the long terminal repeat (LTR) of RSV (_)and SV40-denved sequences that contain the early region polyade-nylation signal (niI) are indicated. Vector sequences that providethe plasmid origin of replication (ori) and the ampicillin resistancegene (ampR) and unique restriction sites available for cloning are

shown. cD)NA inserts for DRot and DRP were subcloned into RSV.3as indicated. At the 3' end of the cDNA, 35 base pairs of SV40 whichare derived from the cDNA cloning vector (LI) are shown.

pUC12 (25) into HindIII-HpaI-digested pRSV-,-globin (12).Transcripts produced from RSV.3 thus lack a splice site(Fig. 1). RSV.2 was constructed by adding the HindlIl-BamHI linker fragment from pUC12 into HindIII-BglII-digested pRSV-3-globin. Transcripts produced from RSV.2thus contain the simian virus 40 (SV40) small t splice site.Because pilot experiments had demonstrated that cDNAinserts in RSV.3 could be expressed as cell surface proteinsin the absence of a splice site (unpublished data), it was

decided to pursue experiments with RSV.3 rather thanRSV.2 because introns can be aberrantly spliced after trans-fection (21). Full-length cDNA inserts for DRa (32) andDR1B (33) were cloned into RSV.3 as shown in Fig. 1. AcDNA insert for DR4Dw4, (clone II in reference 22) wascloned into RSV.3 as follows. The DR4P cDNA was excisedwith NsiI (treated with S1 nuclease) and Sacl. The last 64base pairs of the 3'-untranslated region after NsiI were thusremoved. Sacl cuts within the DNA encoding the DR,signal sequence. This DR4P insert was cloned into Sacl-HpaI-digested RSV.2DR1P. HpaI-digested RSV.2 loses thesplice site; resulting clones are thus called RSV.3. The5'-untranslated region and the first 23 amino acids of thesignal sequence were derived from DR1f. This strategy ofreplacing the DR1,l insert by the DR4,B insert cut within thesignal sequence had the advantage of removing a longsynthetic GC tract from the original DR4P cDNA clone. TheRSV.2 and RSV.3 vectors, together with their map andsequence, and the complete sequence of the DR4P cDNA(unpublished) are available on request.

Full-length cDNA clones for the matrix (1) and the nucle-ocapsid (29) proteins of measles virus were subcloned in theRSV expression vectors. A 1,323-base-pair Sall-SmaI frag-ment of the matrix cDNA was cloned into SalI-HpaI-digested RSV.2 to generate RSV.3 MV-M. A 1,627-base-pairHgiAl (treated with S1 nuclease)-XbaI fragment of thenucleocapsid cDNA was cloned into Hindlll (filled withKlenow polymerase)-XbaI-digested RSV.3. RSV.3 MV-Mand RSV.3 MV-N were then introduced into vectors provid-ing resistance to hygromycin (41). A map of the resultingconstructs is diagramed in Fig. 2. The RSV.3 MV-M expres-

tk

FIG. 2. Measles virus antigen cDNA expression vector. Thevector was constructed from the PHEBO plasmid and the RSV.3plasmid containing cDNA inserts for measles virus antigens asdescribed in Materials and Methods. Promoter sequences (_)from the thymidine kinase gene (tk) and the RSV long terminalrepeat (LTR), sequences containing polyadenylation signals fromSV40 and from the tk gene (Li), and a fragment derived from theEpstein-Barr virus (EBV) genome ( ) are shown. Cells trans-fected with these vectors can be selected for resistance to hygro-mycin (hygroR).

sion unit was excised with an NdeI (filled) site upstream ofthe Rous sarcoma virus (RSV) promoter and the BamHI sitedownstream of the SV40 termination sequence and wasligated into a HindIll (filled)-BamHI-digested pHEBO vec-tor to produce pHEBO MV-M. The RSV.3 MV-N expres-sion unit was also excised with NdeI (filled) and BamHI(with a partial BamHI digest because of an internal BamHIsite in the nucleocapsid cDNA) and was ligated into Hind(filled)-BamHI-digested pHEBO and p220.2 vectors to pro-duce pHEBO MV-N and p220.2 MV-N, respectively. p220.2(a kind gift of B. Sugden) was derived by the addition of apolylinker in the Narl site of plasmid p201 of Yates et al.(41). pHEBO and p220.2 do not replicate in murine cells butcan be used for stable transfections. For unknown reasons,the level of RNA transcribed from the RSV promoter aftertransfection into L cells of the hygromycin vector was verylow. This low level of expression was observed not only withMV-M and MV-N but also with several other cDNA inserts.

Cells and transfections. The human fibroblast line 637Bwas infected with measles virus as described elsewhere (31).The DAP-3 subline of murine L cells was grown in Dulbeccomodified Eagle medium supplemented with 10% fetal calfserum, 4 mM glutamine, and 10 ,ug of gentamicin per ml.DR-expressing cells were obtained by cotransfecting 10 ,ugof RSV.3DRa, 10 ,ug of RSV.3DR,, and 2 ,ug of pSV2-gpt(28) by the calcium phosphate coprecipitation method (13).Medium containing 6 ,ug (per milliliter) of mycophenolicacid, 15 ,ug of hypoxanthine, and 250 ,ug of xanthine wasadded 48 h after the transfection and was changed every 3days. Cells expressing DR antigen were sorted and cloned asdescribed previously (32). Anti-HLA-DR antibody was ob-tained from Becton Dickinson Immunocytometry Systems,Mountain View, Calif. The HLA class II-positive B-cell line45.1 (18) was used as a positive control for DR expression.Murine DAP-3 cells expressing DR1 or DR4 antigens were

transfected by the calcium phosphate coprecipitationmethod with plasmids pHEBO, pHEBO MV-M, pHEBOMV-N, p220.2, and p220.2MV-N. Clones of hygromycin(350 jig/ml)-resistant DAP-3 cells were assayed for thepresence of matrix or nucleocapsid gene transcripts bydot-blot hybridizations (38). Positive clones were expandedin culture. The presence of MV-M or MV-N proteins intransfected cells was assayed for by immunofluorescence

VOL.. 63, 1989

1758 JACOBSON ET AL.

and immunoprecipitation, using polyclonal antisera andmonoclonal antibodies.RNA hybridization. RNA was extracted from cells, elec-

trophoresed in agarose gels, and transferred and fixed tonylon membranes as described elsewhere (2). The procedureincluded UV cross-linking of the RNA to the membrane,which resulted in greatly enhanced hybridization signalscompared with the 80°C baking step (2).

Oligonucleotides were synthesized on a model 380B syn-thesizer (Applied Biosystems) and were end labeled with[-y-32P]ATP and polynucleotide kinase. The DR1l-specificoligonucleotide was 5'-AGATGCATCTTTCCAGCA. TheDR4,B-specific oligonucleotide was 5'-TGTTTAACCTGCTCCAAG. Both 18-mers were hybridized at 42°C in 0.9 MNaCl-10 mM sodium phosphate (pH 7.0-S5 mM EDTA-2%sodium dodecyl sulfate-100-p,g/ml Escherichia coli tRNA.The MV-M and MV-N cDNA fragments described abovewere labeled by the random-priming method (9) with[32P]dATP and [32P]dCTP. To avoid background hybridiza-tion with ribosomal RNA, the MV probes had to be used atvery high hybridization stringencies (8). The MV-M probewas used at 55°C in 50% formamide-0.75 M NaCl-25 mMsodium phosphate (pH 7.0)-S5 mM EDTA-0.02% each bo-vine serum albumin, polyvinylpyrrolidone, and Ficoll-0.2-mg/ml denatured salmon DNA-1% sodium dodecyl sulfate-10% dextran sulfate. The MV-N probe was hybridized in thesame buffer but at 60°C.CTL assays. Measles virus-specific CTL lines were gener-

ated from individuals HLA-DR-typed as either DR1,blank orDR4,5 by repetitive stimulation of peripheral blood lympho-cytes (PBL) with measles virus-infected autologous PBLwhich had been irradiated with 5,000 rads. The cell lineswere cultured in RPMI 1640 supplemented with 5% humanAB serum plus 10% IL-2 (Cellular Products, Inc., Buffalo,N.Y.) and were subcultured once a week. After five suchstimulations, the cell lines were demonstrated to be bothmeasles virus specific and HLA class II restricted by lym-phoproliferative and cytotoxic T-cell assays (data notshown). Transfected murine L cells were used as targets.The cells were labeled with 100 ,uCi of 51Cr (New EnglandNuclear Corp., Boston, Mass.) for 1.5 h, were washed, andwere plated at 5 x 103 cells per well. CTL assays wereperformed as described previously (16). Cytotoxicity assaysin the presence of chloroquine were carried out exactly asdescribed elsewhere (27, 31). Cocultures were carried out for24 h with 106 unlabeled cells and 106 51Cr-labeled cells. Themixed cells were used as targets in a 5-h CTL assay with 104cells per well and at the indicated effector-to-target ratios.

RESULTS AND DISCUSSION

In previous experiments, we had shown that a humanfibroblast line transfected with expressible HLA-DR cx and 1cDNAs and infected with measles virus was lysed by mea-sles virus-specific DR-restricted CTL (31). The expression ofDR antigen at the surface of these transfected human fibro-blasts was low and was not stable over periods of severalweeks. Instability of DR expression in transfected cells wasalso observed in other human cell lines (unpublishedresults). In contrast, the murine fibroblast L-cell line waseasily transfected and it expressed high and stable levels ofsurface DR antigens. We chose to exploit the efficienttransfection of murine L cells, in combination with a strongpromoter for cDNA expression, to produce, by sequentialtransfections, cells expressing human class II antigens andspecific measles virus antigens.

i15

A I

I,

.I

EE IjI5''_

°~ I'-f T T¾ ln A

I\'

a" 101 10 10' I20 10'

Fluorescence intensity

DAP-3

DAP-3DR1

DAP-3DR4

B-LCL

FIG. 3. HLA-DR expression in transfected murine fibroblasts.Cells were stained with a fluorescein isothiocyanate-labeled goatanti-mouse immunoglobulin antibody for control (---) and with afluorescein isothiocyanate-labeled anti-HLA-DR antibody (-).(a) Untransfected DAP-3 cells. (b) DAP-3 cells transfected withRSV.3DRa, RSV.3DR1,B, and pSV2-gpt. (c) DAP-3 cells trans-fected with RSV.3DRot, RSV.3DR4P, and pSV2-gpt. (d) HumanB-cell line 45.1.

Expression of HLA-DR on transfected murine fibroblasts.The long terminaf repeat of RSV is one of the strongestknown promoters (11). cDNA clones for the alpha and betachains of human HLA-DR antigens were inserted into anexpression vector with the RSV long terminal repeat as thepromoter (Fig. 1). The small splice site present on theoriginal pRSV-1-globin vector (12) was found to be dispens-able for surface DR expression both in transient and in stabletransfection experiments (data not shown). To produce cellsexpressing DR1 or DR4 antigens at their surface, RSV.3DRotwas cotransfected with either RSV.3DR1P or RSV.3DR4Ptogether with the pSV2-gpt plasmid for selection. After beingselected with mycophenolic acid, cells were sorted for DRexpression and were cloned. The level of DR antigen de-tected by fluorescein isothiocyanate-labeled antibody oncloned transfectants was homogeneous and was six timesless (for DR1) and three times less (for DR4) than the levelpresent in an Epstein-Barr virus-transformed human B-cellline (Fig. 3). DR expression on these transfectants has been

J. VIROL.

HLA-DR-RESTRICTED PRESENTATION OF CYTOPLASMIC ANTIGENS 1759

b 637B

Pr obe

637B-MV

:0 0 0

DAP-3z E: zI

-tfY

zr Zr> > >

DRI MV-M i

DR4 MV-N V

FIG. 4. Transcription of HLA-DR and of measles virus genes intransfected murine DAP-3 fibroblasts. (a) A 2.5-,ug sample of totalRNA from the indicated cells was size fractionated in a formalde-hyde agarose gel, transferred to a charged nylon membrane, andhybridized with oligonucleotides specific for the DR1 or the DR4,-chain-coding sequence. A DR1 hemizygous B-lymphoblastoid cellline (lane B-LCL DR1) and a DR4,6 heterozygous line (lane B-LCLDR4,6) were used as positive controls for the respective DR,mRNAs. The DR1lB transcription unit in transfected cells is about240 bases longer than that of DR41 because it contains additionalvector sequences. (b) A 10-,ug sample of total RNA from theindicated cells was size fractionated in formaldehyde agarose gels,transferred to charged nylon membranes, and hybridized withmeasles virus matrix (MV-M) or measles virus nucleocapsid (MV-N) gene fragments. 637B is a human fibroblast line which can beinfected in vitro with measles virus. RNA prepared from measlesvirus-infected 637 (lanes 637B + MV) was diluted to 10-2, 10-3, and10-4 into RNA from uninfected 637B. The major measles virusmatrix and nucleocapsid transcripts in infected cells are 1,470 and1,693 nucleotides long, respectively (8). Murine DAP-3 cells ex-pressing DR1 or DR4 antigens were transfected with vectors carry-ing expressible measles virus matrix or nucleocapsid genes (lanesDR1 MV-N, DR4 MV-M, and DR4 MV-N). Arrowheads indicatethe positions of measles virus-specific transcripts in the transfectedDAP-3 cells. These transcripts are longer than in infected cellsbecause they contain additional vector sequences. The upper banddetected with the MV-N probe in the transfected cells is not MV-Nspecific and may represent contamination of the MV-N probe byvector sequences.

stable for months. Because of the lack of monoclonal anti-body specific for the DR1 or DR4 molecule, syntheticoligonucleotides were used to show that the transfected cellsexpressed RNA corresponding to DR1l3 or DR4,, respec-tively (Fig. 4a).

Expression of measles virus matrix and nucleocapsid genesin transfected murine fibroblasts. The measles virus matrixand nucleocapsid proteins were chosen to test whethercytoplasmic proteins could be involved in class 1I-restrictedpresentation to measles virus-specific CTL. cDNA clonesfor MV-M and MV-N were inserted into the RSV.3 expres-sion vector. The RSV.3 MV-M and RSV.3 MV-N expressionunits were subsequently inserted into vectors carrying thehygromycin resistance gene (Fig. 2). DR1- and DR4-positivemurine L cells were transfected with these vectors and wereselected for hygromycin resistance. The expression of sur-face DR antigen on L cells was unaffected by the transfec-tions with hygromycin resistance vectors (data not shown).The level of MV-M or MV-N RNA expressed in clonedtransfectants was determined by RNA blot hybridizationsand was quantitated by comparison with serial dilutions ofRNA extracted from a measles virus-infected cell line (Fig.

40

30

- 20'

_- 10_e

(i,ct

DR1 DR4C MV-N C MV-N

A B C D

.-.,-

25 10 4040

30

20

10

2 5 10 40 2 5 10 40 2 5 10 40

CTL TO TARGET RATIO

FIG. 5. HLA class II-restricted lysis of murine fibroblasts ex-

pressing the measles virus nucleocapsid gene by measles virus-specific cytotoxic T-cell lines. DR1- and DR4-positive murine DAP-3 fibroblasts were transfected either with the hygromycin resistancevector as a control (C) or with the same vector carrying an

expressible measles virus nucleocapsid gene (MV-N). Effector cellswere either a DR1-restricted (A to D) or a DR4-restricted (E to G)measles virus-specific CTL line. The data are presented as percentspecific lysis versus CTL-to-target ratio. Spontaneous release from51Cr-labeled targets was less than 10%. Control experiments withunstimulated PBL gave background levels of lysis (results notshown). Control experiments with autologous B-cell lines infectedwith measles virus gave levels of lysis very similar to those obtainedwith the transfectants (data not shown).

4b). MV-M transcripts were about 10-3 of the level found ininfected cells, and MV-N transcripts were only 1o-4 to 10-3of the level found in infected cells. The matrix and nucleo-capsid proteins were undetectable in the transfectants byimmunofluorescence or by immunoprecipitation of cellspulse-labeled with [35S]methionine (data not shown). Thelack of detection could be due to the low level of expression,to degradation in the cytoplasm, or both.

Measles virus-specific CTL lyse murine fibroblasts express-ing cytoplasmic viral antigens. Measles virus-specific classIT-restricted CTL lines were obtained by repeated in vitrostimulation of PBL with measles virus-infected cells. De-spite the very low level of MV-N transcripts in the trans-fected murine cells and the lack of detectable nucleocapsidprotein, both the DR1- and the DR4-positive cells were lysedby specific CTL lines in a class II-restricted manner (Fig. 5).L cells expressing DR4 and MV-M were also killed by a

measles virus-specific T-cell line (Fig. 6). The MV-M- andMV-N-expressing L cells were lysed as efficiently as a

measles virus-infected autologous B-cell line (data notshown). The efficient class II-restricted CTL recognition oftransfected target cells expressing undetectable amounts ofviral antigen is similar to results obtained by Townsend et al.(35) with class I-restricted recognition of murine L cellstransfected with the influenza virus nucleocapsid gene.

Internal measles virus proteins which are not expressed on

the host cell membrane during virus maturation can thus berecognized by CTL. This component of the cellular immuneresponse to measles virus may be important in normallong-term immunity since it provides a means of eliminationof persistent virus before maturation and release of infec-tious virus. It has been proposed that class IT-restricted CTLmay have important regulatory functions (3). It is possiblethat the same cell that is capable of lysing a target cell

a

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TRANSFECTED TARGETS

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VOL. 63, 1989

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1760 JACOBSON ET AL.

~j30

20

CL.

In

be

S1Cr-labeledunlabeled

2.510 20 2.510 20 2.510 20 2.5 10 20

CTL TO TARGET RATIOFIG. 6. Coculture of target cells. DR4-positive murine fibro-

blasts were transfected with the hygromycin resistance vector aloneas a control (C) or with the same vector carrying an expressiblemeasles virus matrix gene (MV-M). "Cr-labeled target cells were

cultured for 24 h prior to the CTL assay either in the absence (-) or

presence of unlabeled cells as indicated. Effector cells were a

DR4-restricted measles virus-specific CTL line.

expressing measles virus protein epitopes in conjunctionwith class II HLA molecules can provide help for theproduction of measles virus antibody. This would result inongoing antibody production but elimination of infectedantigen-presenting cells before release of infectious virus.

Importantly, recognition of internal components of thevirus may also contribute to diseases caused by measlesvirus, such as subacute sclerosing panencephalitis (SSPE).Although only small amounts of RNA transcripts encodingviral surface antigens are found in brains with SSPE, normalamounts of transcripts encoding nucleocapsid are present (7)and could allow CTL recognition of the infected cells. Thiscould be particularly true if the virus is able to enhance theexpression of class II molecules on glial cells (23). Areduction in measles virus-specific CTL in the peripheralblood of patients with SSPE has been demonstrated recently(S. Dhib-Jalbut, S. Jacobson, D. E. McFarlin, and H. F.McFarland, Ann. Neurol., in press). One of the possibleinterpretations of this finding is that these cells are seques-

tered within the brain and contribute to disease by recogni-tion and lysis of the infected cells. The recognition of MV-M-or MV-N-transfected cells that do not express antigendetectable by antibody also raises the possibility that recog-

nition of viral components by CTL could contribute todiseases in which viral antigens are not readily demon-strated. An example is multiple sclerosis. The measles virusgenome has been reported to be present in brain tissue frompatients with and without multiple sclerosis, although viralantigen was undetectable (14). The level of measles virus-specific CTL was reduced in some patients with multiplesclerosis (17), which again raises the possibility of seques-

tration of a CTL population recognizing epitopes not demon-strable by anti-measles virus antibody. As with the SSPE,this hypothesis is speculative. Examination of these possi-bilities requires a better understanding of the regulation ofclass II molecule expression on cells of the nervous system

and an examination of the relevant CTL populations, partic-ularly those found within the nervous system in thesediseases.

Processing of measles virus cytoplasmic antigens. ClassIT-restricted CTL most likely recognize processed viralpeptides, as do class II-restricted helper cells (20) and classI-restricted CTL (37). Chloroquine inhibition of class II-restricted CTL recognition (10, 27) supports this interpreta-tion and suggests that processing occurs in endosomes. TheCTL recognition of MV-M and MV-N transfectants demon-strates that endogenously synthesized proteins can be pre-

sented by class II MHC antigens. Several pathways ofantigen processing may operate in this case. The first possi-bility is that endogenously synthesized proteins are releasedfrom the cells and that processing follows the exogenous-endocytic route typical of antigen-presenting cells. Thesecond possibility is that endogenous processing targetscytoplasmic antigens to endosomes. Finally, the processingof cytoplasmic antigens for class II-restricted presentationmay follow the same pathway as that used for class I-

restricted presentation. None of these possibilities has beenruled out, but the following experiments were carried out toaddress this issue.

Cell-mixing experiments were carried out to test whetherproteins synthesized in the cytoplasm exit the cell andreenter by endocytosis to associate with class II antigens(Fig. 6). 51Cr-labeled cells were incubated for 24 h prior tothe CTL assay. Labeled DR4 MV-M-transfected cells were

still lysed after a 24-h incubation in the absence or presenceof unlabeled DR4-transfected cells. However, labeled DR4-transfected cells were not lysed above background levelsafter incubation with unlabeled DR4 MV-M-expressing cells.This result argues against an exogenous pathway in thepresentation of MV-M antigens by transfected cells but doesnot rule out the possibility of antigen recycling from the cellsurface. It is difficult to interpret chloroquine inhibition ofpresentation by stably transfected cells because peptide-class II antigen complexes are known to be stable (6). It ispossible that preexisting complexes remain at the cell sur-

face throughout treatment with chloroquine. Nevertheless, itis worth noting that lysis of the transfected cells by classII-restricted CTL was totally unaffected by a 12-h treatmentwith chloroquine (data not shown). Similarly, chloroquinetreatment of target cells prior to and during infection withmeasles virus had no effect on the recognition and lysis byclass TI-restricted CTL even though, under identical condi-tions, lysis of influenza virus-infected cells was completelyabolished (31).Murine L cells, such as those used in this study, express

only a small amount of invariant chain (19; unpublishedobservation). In normal antigen-presenting cells, the invari-ant chain is associated intracellularly with class II antigensand is synthesized in excess over the alpha and beta chainsof class II molecules. The presence in the transfected targetcells of human class II molecules that are not complexedwith the invariant chain provides an interesting interpreta-tion. The role of the invariant chain may be to preventbinding of peptides to class II molecules until they reachendosomes (21a). As a result, transfected cells expressing no

detectable invariant chain (31) or low levels of invariantchain may be permissive to the presentation of endogenousantigens by class II molecules. This hypothesis is stillspeculative but reconciles data presented here with thedistinct processing requirements for class I- and class II-

restricted antigen presentation (27). The distinction betweenclass I- and class II-restricted presentation may not be basedonly on separate processing pathways but also on differentaccessibilities of the class I and class II peptide-bindingsites. Alternative interpretations can also be formulated on

the basis of the different modes of entry used by influenzavirus and measles virus (21a).The elucidation of processing pathways for class I- and

class II-restricted presentation of cytoplasmic antigensawaits further studies. The main conclusion from this studyis that cytoplasmic measles virus proteins can be presentedto CTL by MHC class II antigens.

DR4 TRANSFECTED TARGETSC |MV-M C |MV-M- - MV-M C

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HLA-DR-RESTRICTED PRESENTATION OF CYTOPLASMIC ANTIGENS 1761

ACKNOWLEDGMENTS

We thank W. Bellini, B. Howard. and B. Sugden for gifts ofplasmids and G. Shaw for her kind editorial assistance.

LITERATURE CITED

1. Bellini, W. J., G. Englund, C. D. Richardson, S. Rozenblatt, andR. A. Lazzarini. 1986. Matrix genes of measles virus and canine

distemper virus: cloning, nucleotide sequences. and deducedamino acid sequences. J. Virol. 58:408-416.

2. Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E. 0. Long.1988. CD3-negative lymphokine-activated cytotoxic cells ex-

press the CD3F gene. J. Immunol. 140:1685-1689.3. Braakman, E., F. T. M. Botteveel, G. Van Bleek, G. A. Van

Seventer, and C. J. Lucas. 1987. Are MHC class restrictedcytotoxic T lymphocytes important? Immunol. Today 8:265-267.

4. Braciale, T. J., V. L. Braciale, M. Winkler, I. Stroyonowski, L.Hood, J. Sambrook, and M. J. Gething. 1987. On the role of thetransmembrane anchor sequence of influenza hemagglutinin intarget cell recognition by class I MHC-restricted hemagglutinin-specific cytolytic T lymphocytes. J. Exp. Med. 166:678-692.

5. Braciale, T. J., L. A. Morrison, M. T. Sweetser, J. Sambrook,M. J. Gething, and V. L. Braciale. 1987. Antigen presentationpathways to class I and class 11 MHC-restricted T lymphocytes.Immunol. Rev. 98:95-114.

6. Buus, S., A. Sette, S. M. Colon, D. M. Jenis, and H. M. Grey.1986. Isolation and characterization of antigen-la complexesinvolved in T cell recognition. Cell 47:1071-1077.

7. Cattaneo, R., G. Rebmann, K. Baczko, V. ter Meulen, and M. A.Billeter. 1987. Altered ratios of measles virus transcripts in

diseased human brains. Virology 160:523-526.8. Cattaneo, R., G. Rebmann, A. Schmid, K. Baczko, V. ter

Meulen, and M. A. Billeter. 1987. Altered transcription of a

defective measles virus genome derived from a diseased humanbrain. EMBO J. 6:681-688.

9. Feinberg, A. P., and B. Vogelstein. 1983. A technique forradiolabeling DNA restriction endonuclease fragments to highspecific activity. Anal. Biochem. 132:6-13.

10. Fleischer, B., H. Becht, and R. Rott. 1985. Recognition of viralantigens by human influenza A virus-specific T lymphocyteclones. J. Immunol. 135:2800-2804.

11. Gorman, C. M., G. T. Merlino, M. C. Willingham, I. Pastan,and B. H. Howard. 1982. The Rous sarcoma virus long terminalrepeat is a strong promoter when introduced into a variety ofeukaryotic cells by DNA-mediated transfection. Proc. Natl.Acad. Sci. USA 79:6777-6781.

12. Gorman, C., R. Padmanabhan, and B. H. Howard. 1983. Highefficiency DNA-mediated transformation of primate cells. Sci-ence 221:551-553.

13. Graham, F. L., and A. J. Van der Eb. 1973. A new technique forthe assay of infectivity of human adenovirus 5 DNA. Virology52:456-467.

14. Hasse, A. T., L. Stowring, and P. Ventura. 1984. Detection byhybridization of viral infection of the human central nervous

system. Ann. N.Y. Acad. Sci. 229:282-284.15. Jacobson, S., and W. E. Biddison. 1986. Virus-specific HLA

class II-restricted cytotoxic T cells, p. 187-192. In M. B. A.Oldstone and A. Notkins (ed.). Concepts in viral pathogenesisIl. Springer-Verlag, New York.

16. Jacobson, S., J. R. Richert, W. E. Biddison, A. Satinsky, R. J.Hartzmann, and H. F. McFarland. 1984. Measles virus specificT4+ human cytotoxic T-cell clones are restricted by class IlHLA antigens. J. Immunol. 133:754-757.

17. Jacobson, S. J., M. L. Flerlage, and H. F. McFarland. 1985.Impaired measles virus specific T cell responses in multiplesclerosis. J. Exp. Med. 162:839-850.

18. Kavathas, P., F. H. Bach, and R. DeMars. 1980. Gammaray-induced loss of expression of HLA and glyoxalase I allelesin lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 77:4251-4255.

19. Koch, H., and A. W. Harris. 1984. Differential expression of theinvariant chain in mouse tumor cells: relationship to B lymphoiddevelopment. J. Immunol. 132:12-15.

20. Lamb, J. R., D. D. Eckels, P. Lake, J. N. Woody, and N. Green.1982. Human T-cell clones recognize chemically synthesizedpeptides of influenza haemagglutinin. Nature (London) 300:66-68.

21. Laub, O., and W. J. Rutter. 1983. Expression of the humaninsulin gene and cDNA in a heterologous mammalian system. J.Biol. Chem. 258:6043-6050.

21a.Long, E. O., and S. Jacobson. 1989. Pathways of viral antigenprocessing and presentation to cytotoxic T lymphocytes: de-fined by the mode of virus entry? Immunol. Today 10:45-48.

22. Long, E. O., C. T. Wake, J. Gorski, and B. Mach. 1983.Complete sequence of an HLA-DR P chain deduced from a

cDNA clone and identification of multiple non-allelic DR ,Bchain genes. EMBO J. 2:389-394.

23. Massa, P. T., A. Schimpl, E. Wecker, and V. ter Meulen. 1987.Tumor necrosis factor amplifies measles virus-mediated la in-duction on astrocytes. Proc. Natl. Acad. Sci. USA 84:7242-7245.

24. McMichael, A. J., C. A. Michie, F. M. Gotch, G. L. Smith, andB. Moss. 1986. Recognition of influenza A virus nucleoproteinby human cytotoxic T lymphocytes. J. Gen. Virol. 67:719-730.

25. Messing, J. 1983. New M13 vectors for cloning. MethodsEnzymol. 101:20-78.

26. Meuer, S. C., J. C. Hodgdon, D. A. Cooper, R. E. Hussey, K. A.Fitzgerald, S. F. Schlossman, and E. L. Reinherz. 1983. Humancytotoxic T cell clones directed at autologous virus transformedtargets: further evidence for linkage of genetic restriction to T4and T8 surface glycoproteins. J. Immunol. 131:186-192.

27. Morrison, L. A., A. E. Lukacher, V. L. Braciale, D. P. Fan, andT. J. Braciale. 1986. Differences in antigen presentation of MHCclass I- and class Il-restricted influenza virus-specific cytolyticT lymphocyte clones. J. Exp. Med. 163:903-921.

28. Mulligan, R. C., and P. Berg. 1981. Selection for animal cellsthat express the Eschlerichlia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA78:2072-2076.

29. Rosenblatt, S., 0. Eizenberg, R. Ben-Levy, V. Lavie, and W. J.Bellini. 1985. Sequence homology within the morbilliviruses. J.Virol. 53:684-690.

30. Schmid, D. S. 1988. The human MHC-restricted cellular re-

sponse to herpes simplex virus type 1 is mediated by CD4',CD8- T cells and is restricted to the DR region of the MHCcomplex. J. Immunol. 140:3610-3616.

31. Sekaly, R. P., S. Jacobson, J. R. Richert, C. Tonnelle, H. F.McFarland, and E. 0. Long. 1988. Antigen presentation to HLAclass 11-restricted measles virus-specific T-cell clones can occur

in the absence of the invariant chain. Proc. Natl. Acad. Sci.USA 85:1209-1212.

32. Sekaly, R. P., C. Tonnelle, M. Strubin, B. Mach, and E. 0.Long. 1986. Cell surface expression of class 11 histocompatibil-ity antigens occurs in the absence of the invariant chain. J. Exp.Med. 164:1490-1504.

33. Tonnelle, C., R. DeMars, and E. 0. Long. 1985. DO,B: a new Pchain gene in HLA-D with a distinct regulation of expression.EMBO J. 4:2839-2847.

34. Townsend, A. R. M. 1987. Recognition of influenza virus pro-teins by cytotoxic T lymphocytes. Immunol. Res. 6:80-100.

35. Townsend, A. R. M., F. M. Gotch, and J. Davey. 1985. Cyto-toxic T cells recognize fragments of influenza nucleoprotein.Cell 42:457-467.

36. Townsend, A. R. M., A. J. McMichael, N. P. Carter, J. A.Huddleston, and G. G. Brownlee. 1984. Cytotoxic T cell recog-nition of the influenza nucleoprotein and hemagglutinin ex-

pressed in transfected mouse L cells. Cell 39:13-25.37. Townsend, A. R. M., J. Rothbard, F. M. Gotch, G. Bahadur, D.

Wraith, and A. J. McMichael. 1986. The epitopes of influenzanucleoprotein recognized by cytotoxic T lymphocytes can bedefined with short synthetic peptides. Cell 44:959-968.

38. White, B. A., and F. C. Bancroft. 1982. Cytoplasmic dot

VOL. 63. 1989

1762 JACOBSON ET AL.

hybridization. Simple analysis of relative mRNA levels inmultiple small cell or tissue samples. J. Biol. Chem. 257:8569-8572.

39. Wraith, D. C. 1987. The recognition of influenza A virus-infected cells by cytotoxic T lymphocytes. Immunol. Today8:239-246.

40. Yasukawa, M., and J. M. Zarling. 1984. Human cytotoxic T cellclones directed against Herpes simplex virus-infected cells.Lysis restricted by HLA class II MB and DR antigens. J.Immunol. 133:422-427.

41. Yates, J. L., N. Warren, and B. Sugden. 1985. Stable replicationof plasmids derived from Epstein-Barr virus in various mamma-

lian cells. Nature (London) 313:812-815.42. Yewdell, J. W., J. R. Bennink, and Y. Hosaka. 1988. Cells

process exogenous proteins for recognition by cytotoxic Tlymphocytes. Science 239:637-640.

43. Yewdell, J. W., J. R. Bennink, G. L. Smith, and B. Moss. 1985.Influenza A virus nucleoprotein is a major target antigen forcrossreactive anti-influenza A virus cytotoxic T lymphocytes.Proc. Natl. Acad. Sci. USA 82:1785-1789.

44. Zinkernagel, R. M., and P. C. Doherty. 1979. MHC restrictedcytotoxic T-cells: studies on the biological role of polymorphicmajor transplantation antigens determining T-cell restrictionspecificity. Adv. Immunol. 27:51-177.

J. VIROL.