Deficient antigen-presenting cell function in
multiple genetic complementation groups of
type II bare lymphocyte syndrome.
S Kovats, … , W W Kwok, J S Blum
J Clin Invest.
1995;
96(1)
:217-223.
https://doi.org/10.1172/JCI118023
.
The absence of HLA class II gene expression in type II bare lymphocyte syndrome (BLS)
results from defective transcriptional activation of class II histocompatibility genes. Genetic
studies have revealed that distinct defects in multiple trans-acting factors result in the
immunodeficient BLS phenotype. We studied antigen-presenting cell (APC) function in
DR-transfected BLS cells derived from multiple complementation groups. Each BLS cell line
displayed the same defective APC phenotype: an inability to mediate class II-restricted
presentation of exogenous protein antigens, and structurally altered class II alpha beta
dimers. Expression of the HLA class II-like genes DMA and DMB, previously implicated in
antigen presentation, was reduced or absent in the BLS cells. Fusion of BLS cells with cell
line 721.174, which has a genomic deletion of HLA class II genes, coordinately restores
class II structural gene and DM gene expression and a wild-type APC phenotype. Thus
each of the molecular defects that silences class II structural gene transcription also results
in a defective APC phenotype, providing strong evidence for coregulation of these two
functionally linked pathways.
Research Article
Find the latest version:
Deficient Antigen-presenting Cell
Function in
Multiple
Genetic
Complementation Groups
of
Type 11
Bare
Lymphocyte Syndrome
Susan Kovats,* Gerald T.Nepom,* Marilyn Coleman, Nepom,*
Immunology and Diabetes Programs, VirginiaMasonResearch Center, Seattle, Washington 98101;andDepartments of *Immunology andtPediatrics, University of Washington School of Medicine, Seattle, Washington 98195
Abstract
The absence of HLA class II gene expression in
type
II bare lymphocyte syndrome (BLS) results from defectivetranscriptional activation of class II histocompatibility
genes.Genetic studieshaverevealed that distinct defectsin
multipletrans-acting factors result in theimmunodeficient BLSphenotype. We studiedantigen-presenting cell(APC) functioninDR-transfected BLScellsderived from multiple complementationgroups. Each BLS cell linedisplayed the
samedefective APC phenotype:aninabilitytomediate class
II-restricted presentation ofexogenousprotein antigens,and structurally altered class II a(3 dimers. Expression ofthe HLAclassH-like genes DMA andDMB, previously
impli-catedin antigen presentation,wasreducedorabsentinthe BLScells. Fusionof BLScells with cell line721.174, which has agenomic deletion ofHLA class II genes,coordinately
restores class II structural gene and DM gene expression
andawild-type APC phenotype. Thuseachofthe molecular defects that silences class H structural gene transcription
also results inadefectiveAPCphenotype, providingstrong
evidence for coregulation ofthese two functionally linked
pathways. (J. Clin. Invest. 1995.
96:217-223.)
Key words: geneexpression regulation* genes MHC class H *immuno-competence * disease* HLA-DR
antigen
Introduction
Type H barelymphocyte syndrome
(BLS),'
orHLA class11-deficientcombined immunodeficiency, isan autosomal
reces-sive disorder in which the HLA class II structural genesare not
transcribed in any tissue either constitutively or in response
to inducing stimuli (1, 2). The resulting absence of class H
histocompatibility antigen expression on antigen-presenting
cells, suchas B lymphocytes and cells of the macrophage
lin-Address correspondence to Susan Kovats, Virginia Mason Research
Center, 1000 SenecaSt., Seattle, WA 98101. Phone: 206-223-8813;
FAX:206-223-7543.J. S. Blum's currentaddress isDept.of
Microbiol-ogy andImmunology, Indiana UniversitySchoolof Medicine, Indianap-olis,IN46202.
Receivedforpublication 26 January1995 andacceptedinrevised form24 March 1995.
1.Abbreviations usedinthispaper: APC, antigen-presentingcells;
B-LCL, B lymphoblastoid cell line; BLS, bare lymphocyte syndrome;
CLIP,invariant chain-derivedpeptide; HA,influenzahemaglutinin;TT,
tetanustoxoid.
eage, significantly impairs cellular and humoral immune re-sponses (reviewed in reference 3). Thus, BLS immunodefi-ciency is characterized byapaucity of T lymphocyte responses andimpairedantibody productionuponchallengewith foreign antigens, leading to extreme susceptibility to viral, bacterial,
andfungal infections. Thesepathophysiologic defects in BLS have beendirectly attributedto alack of HLA class H-restricted antigen presentation to T lymphocytes.
Theabsence of HLA class H gene expression results from mutations in transactivator genes necessarytomediate transcrip-tion of thecoordinately regulated class H genes (2, 4, 5). Ge-netic studies have revealed that at least four distinct molecular defects manifest as the class II null phenotype of BLS patient cells(6, 7). We havepreviouslydemonstrated thataBLS cell line(BLS-1 ) isolated fromapatientfromoneof these comple-mentation groups, following transfection with wild-type class H genes, hasdefects in class11-mediatedpresentation of native exogenous antigens and structurally altered class H af3 mole-cules (8). This finding suggested that the molecular pathways regulating HLA class H structural gene expression and those
modulatingpathways of class II functional antigenprocessing
andpresentation sharecommonelements, possibly coregulated by the transcriptionally active elements missing in patients
with BLS.
Presentation of exogenous foreign antigens by
antigen-pres-entingcells(APC)isacomplex process, in whichantigensare
proteolyticallyprocessed to peptide epitopes that are then bound by class H molecules and displayed at the cell surface for recog-nition by antigen-specific CD4+ helper T lymphocytes (re-viewed in reference 9). Mutational analyses have shown that genes essential forappropriate class11-mediatedantigen
presen-tation, recentlyidentifiedas HLA-DMA and -DMB (10, 11), map withinthe class
HI
region of the MHC (12-15), raising thepossibility that thisclusteringof genes encoding both class H structural proteins and accessory molecules required for theirantigen-presentingfunction wouldensuretheir coordinate regu-lation. In addition, there may be multiple genes outside the MHC that arerequired for optimal antigen presentation, such
astheinvariant chain ( 16), that share regulatory elements with HLAclass II genes.
To analyze the coregulation of these phenotypes, class H transcription and antigen presentation, we transfected class H HLA-DRmolecules intodifferentBLScell lines, thereby restor-ingstructuralgeneexpression.Several structural andfunctional aspects of class I-dependentantigen processing and presenta-tionwere thentestedinthesecells.DR-transfectedBcelllines,
BLS-1, SJO, and 6.1.6, representative of three distinct BLS complementation groups, showedthesamedefective APC
phe-notype:(a)inabilitytoparticipate in the class H-mediated pre-sentation of exogenousnativeproteinantigensalthough presen-tation of preprocessed peptideswasefficient; and (b)
structur-allyabnormal class II ac dimers with reduced stability in SDS. The distinct transactivators defective in each of the different J.Clin. Invest.
)The AmericanSocietyforClinicalInvestigation,Inc.
BLS groups were required for expression of genes involved in
class II-mediated antigen presentation, indicating a common
transcriptional pathway coordinate withstructural class IIgene
expression.
Methods
Cell lines.BLS-land BLS-2 (17) (kindly provided by J. Lee, Memorial
SloanKettering Cancer Center, New York), and SJO (18) are HLA class11-nullB-LCLgenerated from cells of patients with type II Bare Lymphocyte Syndrome and are members of complementation groups I, II, and IV, respectively (7). The class II-nullB-LCL 6.1.6 was generated after in vitro mutagenesis (19). 6.1.6 (classified as complementation group III[7])has a mutation resulting in the absence of class II tran-scription and thus exhibits the class II null phenotype of BLS patient cells. T2(.174 x CEM.T2) is a human cell hybrid that lacks all four copies of the MHC class II region on chromosome 6 (20). Class II DRa(DRAl)and
DR,/
(DRB1*0401) cDNA encoding DR4w4mole-cules were introduced into SJO and 6.1.6 using retrovirus-mediated gene transfer(21).TheBLS-l.DR4w4, BLS-1.DR3, BLS-L.DR5,and T2.DR4w4 transferent cells have been described (8).BLS-l.DR4w14
transferent cells were generated by introduction of DRAI and
DRB1*0404 genes. 721.174 is a mutant(generatedinvitro)human B cell line that has ahomozygous deletion of the MHC class II region (22); .174 cells containing either neomycin-resistance or gpt (guanine
phosphoribosyltransferase) genes were kindly providedby R. Green-wood and R. DeMars (University ofWisconsin, Madison, WI). The DR4w4 B cell lines Priess and Savc, and DR3 B cell lines Mat and Vavy wereused as wild-type controls. Cell lines were grown in
RPMI-1640mediumplus 10% FCS(Hyclone,Logan, UT).
Antibody bindingassays. mAb bindingto live cells was detected
byflow cytometryusingaFACScan(BectonDickinson andCo.,
Cock-eysville, MD).Theanti-DR mAb L243(23)and anti-DR3 mAb 16.23
(24) wereused atsaturating concentrationsanddetectedusing
FITC-conjugatedgoat anti-mouseIg (JacksonImmunoResearch Labs.Inc.,
WestGrove,PA).
Assays with T cellhybridomas. Antigen-specificTcellhybridomas
weregenerously supplied byP.Whiteley (MerckResearchLabs., Rah-way,NJ). TheDR4w4-tetanus-specificTcellhybridoma49.23.2was generated after tetanus immunization ofa DR4w4 transgenic mouse
(25). Formalin-fixedtetanustoxoid (Ti')(Wyeth Laboratories,
Phila-delphia, PA)wasdigestedwith N-tosyl-L-phenylalanine chloromethyl
ketonetrypsin.The presence ofantigenic peptidesinthetryptic digest
of TTwasconfirmedbyincubatingthedigested preparationwith
para-formaldehyde-fixed APCs. APC(105), Ti antigen (0.4 ,uMofnative
ortrypsin-digested),and49.23.2 T cells(105)wereincubatedtogether
for 24 h. Insomeexperiments,APCwerepreincubatedwitheither 0.4
or0.8 /Mof TTantigen(nativeortrypsin digested) for 3 h at37°C
and subsequently washed twice with medium beforeplating in wells with T cells.
TheDR4w4-hemagglutinin-specificTcellhybridoma50.84.17was generatedafterimmunization ofaDR4w4transgenicmousewith fixed
influenzavirus;the Tcellhybridomaisspecificfor the influenza
hemag-glutinin peptide epitope HA 307-319 (PKYVKQNTLKLAT). APC (105) wereincubated with either 1.0
MM
HA 307-319peptide or50Mg/ml
fixedA/Mississippi/1/85 (H3N2) influenza virus(Connaught Laboratories, Swiftwater,PA) for 3 hat37°Candsubsequentlywashed twice with medium beforeplatinginwells with 50.84.17Tcells(105).T cell IL-2 and IL-4 production was determined by survival of an
IL-2/IL-4-dependent cell line, HT-2, measured using [3H]thymidine incorporation (0.5 ,uCi/well) (26). The T cell hybridomas did not
produce lymphokines in response toAPCs in the absenceofantigen.
Assayswererepeatedthreetofour times with similar results. mRNA analyses. RNA was isolated from 5-10 x 106 cells, and cDNA was made from 1.0 Mg ofRNA using reverse transcriptase.
Primer-directedamplification of cDNAwasaccomplished usingPCR. 5' and3' primersforamplificationofDMA cDNAwere:5 '-ACiTITI'-CCCAGAACACTCGGand S'-CTGGAAGCTGAGTCCATCG and the
expected product is 340 bp. 5' and 3' primers for amplification of DMB cDNAwere: 5'-ACAGCACCTCAACCAAAAAGA and 5 '-GGGGTT-AAGGCTAAATGGGA and the expected product is 320 bp. DMA and DMB cDNA were amplified for 30 cycles (denaturation for 1 min at 930C, annealing for 1 min at 550C and polymerization for 1 min at
720C).The reaction products were visualized by agarose gel electropho-resis andstaining with ethidium bromide. PCR products were sized using
a 1-kb DNA ladder (GIBCO BRL, Gaithersburg, MD). The identity of theputative DMAband amplified inBLS-l wasconfirmed by DNA sequence analysis. The presence of intact cDNA in samples derived from cellular RNA was confirmed by the presence of a product amplified using primers specific for the actin gene. A higher molecular weight bandamplified in someofthe RNA samples is likely to be resulting fromgenomicDNAcontamination of the RNA sample, since the size of theproduct(- 1 kb for DMA and- 1.5kb for DMB) corresponds
to the size of the product directly amplified from genomic DNA of Vavy.
Cellfusions.Fusions between BLS-l (DR3,5)and .174 or between SJO (DR5,7) and .174 were accomplished using 50% polyethylene
glycol-1500,5% DMSO (4). Before cell hybridization, genes encoding
drug resistance were' introduced into the cell lines as follows: BLS-1,
hygromycin-resistance gene; .174, neomycin-resistance orgpt genes; andSJO,neomycin-resistancegene linked to a DRB1 *0401 gene. The
BLS-l x .174 (neo) fused cells were selected using hygromycin B
(Calbiochem Novabiochem, La Jolla, CA) (150
pg/ml)
and G418(GIBCO BRL) (1 mg/ml). The SJO x .174 (gpt) fused cells were selected forusingG418 and mycophenolic acid (10
pg/ml)
plus xan-thine (6Ag/ml)
(Sigma Immunochemicals, St. Louis, MO). Stableindependentlyfused drug-resistant populations were analyzed. The BLS-1 x SJOhybridshavebeen described (8).
Analyses ofHLAclass II dimers using immunoblotting. Cells were lysedat 5 x 107/mlin 1% NP-40,0.15M NaCl,5mMEDTA, 50mM Tris-Cl,pH 7.2, 1.0 mM PMSF, 1.0ag/mlleupeptin, and 1.0
/Lg/ml
pepstatin for 30 min on ice; protease inhibitors were obtained from
Boehringer Mannheim Corp. (Indianapolis, IN). To detect HLA-DR
af dimers,50 ,Igof total cell lysate wasmixed with the appropriate volumeof 4xnonreducingSDSsamplebuffer(lx concentration: 62.5 mMTris-Cl,pH6.8,0.2% SDS, 10% glycerol) and incubated for 30 minat room temperature. To detect
HLA-DRP3
monomers, 25 ,ug of total celllysatewasmixed withanappropriatevolume of 4xreducingSDS sample buffer (lx concentration asabove plus 100 mM DTT) and boiled for 10 min.Sampleswereelectrophoresedona 10%
SDS-polyacrylamide gelandsubsequentlytransferredtonitrocellulose.
Im-munoblotting was accomplished using hybridoma supernatant
con-taining mAbsL243(23)orXD5.Al 1 (27). BindingofprimarymAbs
wasdetectedusing peroxidase-conjugatedF(ab')2goat anti-mouse IgG
+ IgM (JacksonImmunoResearchLaboratories)anda
chemilumines-cent substrate for peroxidase, ECL, (Amersham Corp., Arlington
Heights, IL)followedbyexposuretofilm.
Results
HLA-DRtransfectedBLScells exhibitconformationallyaltered
class II dimers. Defects in class II antigenstructurehave been linked with theimpaired ability of bothinvitro-generated ( 11, 13, 14, 28) and naturally occurring (BLS-1) (8)mutantcells topresentpeptidesderived fromexogenously supplied antigens.
Previously we showed that BLS-1 cells expressing introduced DR4w4 and DR3 molecules failed tobind allele-specific anti-DR3 and -DR4w4 mAbs that recognize
conformation-depen-dent epitopes on these DR molecules (8). These
conforma-tion-dependentepitopes apparentlyreflectthespecificityof se-lectedantibodiestorecognizeintactpeptide/classTIcomplexes.
Structurally altered peptide/class II complexes also show a markedreduction in ability toremain stable DR
a,/
heterodi-mers after roomtemperature incubation in low concentrations
~~~~~~iq
3 3t B
3:
Wt 3: 0 q v
cn a CC a a a a
Cl) *o a InUr cn cn :
r 0 m m m m
V- I m
W R
UL LLI
O r r% t t
cn
' N Nx x x '
-' '- - O c
0 c>
i
-Figure1. HLA-DR transfectedBLS cells contain
DRac/
conformationally
altered classIIa,(
dimers thatareunstable inSDS,and are renderedSDS-stableupon cell fusion. Top, detectionof 60kD class11 af3 dimers in unboiled celllysates in 0.2% SDSby immunoblottingwith mAbL243.Botton, detection of the 28kDDRl6chain inboiledandreduced cell
DR/? lysates in 0.2% SDS by immunoblotting with mAb XD5.A 1.Namesof cell lines in each lane are de-picted above both panels. Twoindependently fused
BLS-l x .174 orSJO x .174hybridsareshown.
Toinvestigate if abnormally conformed class11 molecules are characteristic of different BLS complementation groups, genes encoding DRa (DRA1) and
DR4w4,6
(DRB1*0401) weretransferred into cellsrepresentative oftwoadditional BLS complementation groups, 6.1.6 and SJO. We theninvestigated whetherDRa/3 heterodimers with different allelicspecificities,expressed in distinct BLS cells, would also exhibit this SDS instability. Aliquots of total cell lysatewereincubated in either nonreducing SDSsamplebufferat roomtemperatureorboiled inreducing SDSsamplebuffer before SDS-PAGE and transfer
tonitrocellulose.Immunoblottingwasperformedonthe
nonre-ducedsamplesusingmAbL243(Fig: 1, top) whichrecognizes
mature DR heterodimers, and on the reduced samples using mAb XD5.A11(Fig. 1, bottom), which recognizes DRB
mono-mers.
The DR heterodimers in BLS-1, SJO, and 6.1.6transferents,
detectablebyimmunoblotting with mAb L243, showed greatly reduced stability in SDSascomparedtothewild-type B-LCL Priess and Savc.Noclass II
a,(
dimerwasobserved in BLS-1 cells transfected only with the DRA1 gene. Interestingly, al-thoughtheamountsof DRBmonomer werecomparable in the differentBLS-1transferents,theamountof SDS-stable DRhet-erodimerdetectable variedsignificantly with theDRallele
ex-pressed. DR5 and DR4w14 dimers were more stable than DR4w4dimers, whilenoDR3 dimersweredetected.
The observation of residual stable DR4 and DR5 dimers in BLS cells suggests thebinding ofasubset ofpeptides even in thesemutant APC. In contrast, stable DR3 dimers are not de-tected in BLS cells, aresult also observed in several in vitro
mutantsdefective inantigen presentation (14, 28).DR3
mole-cules in themutantcellsarepreferentially loaded withanested
setofinvariant chain-derived peptides (CLIP)that do not im-part dimerstability in SDS (29, 30). Interestingly, consistent
with our observation, HPLC analyses of peptides eluted from
DR4w4andDR5 moleculespurified from theinvitro-generated
mutantAPCT2.DR4w4andT2.DR5 revealedaminor propor-tionof peptides that weredistinguishable from CLIP peptides andwere notpresent in DR3 molecules purified fromT2cells (31).CLIPbindspromiscuously tomany classIIalleles;
how-ever, its avidity for DR4w4 and DR5 molecules is reduced
compared with DR3 antigens (3 1).Thus, the presence of resid-ual stable DR4 and DR5 dimers in BLS cells may reflect differ-ential CLIP loadingorretentionbyDRmoleculesin these cells aswell aslimited antigenic peptidebinding.
BLScells aredeficient in HLA class II-mediated
presenta-tionof exogenously suppliednativeantigens. Sincestructurally altered class ]I molecules correlate with defects in the peptide-class II complex, theability of DR4w4-transfected BLS cells
tofunction in the class11-mediatedpresentation oftwoforeign
proteinantigens, TT and influenza virus hemagglutinin (HA),
wastheninvestigated (Fig. 2). Upon incubation witha prepara-tion of proteolyzed TT (containing antigenic peptides), BLS-l.DR4w4, 6.1.6.DR4w4, and SJO.DR4w4 cells were able to
stimulatelymphokine production byamurineTcellhybridoma thatisspecificfor TT in thecontextof DR4w4(Fig. 2 A). The
ability of mutant BLS cells to present tetanus peptides was
comparabletothat of thewild-typeDR4w4 B-LCLPriess;the presence ofantigenic peptides in the proteolyzed tetanus was
confirmed by incubation of the preparation with
chemically-fixed Priess cells (datanotshown). In contrast,BLS-l.DR4w4,
SJO.DR4w4, and6.1.6.DR4w4 cells showedamarked reduc-tion in ability to presentpeptide epitopes derived from native TTtothisTcellhybridoma (Fig. 2 A).
Similarly,after incubation with formalin-fixed influenza vi-rus, the three DR-transfected BLS cells(BLS-1, SJO, and6.1.6)
wereeach deficient inpresenting nativeHA to a Tcell hybrid-oma recognizing
DR4w4/HA307-319
complexes (Fig. 2 B). The HApeptide 307-319 was presented with comparable effi-ciency bythe mutant BLSandwild-typePriess cells. The small amount of T cell stimulation observed after incubation of 6.1.6.DR4w4 cells with native TT(Fig.2 A)was notobserved after incubation ofthese cellswith influenzavirus, suggestingthat theseverity of the APCdeficiency may be related to the nature ofthe protein antigen being processed and presented.
Thus, cellsrepresentative of three distinct BLS
complementa-tion groupsweredeficient in the class11-mediated presentation
of the native exogenous antigens, TI and influenza HA,
3~~~
fl~~'-
Y.-V'- cN x x
30.
CO) 0 0 00 N %' JI~ J -,) ;3
.*
aO 1P-
C m m 0 CO)DMA
DMB
Figure 3. BLS cells lack HLA-DM RNA.(Top)PCRamplificationof
cDNAcorrespondingtothe DMA gene; the arrow indicates the expected DMAproduct of340 bp. (Bottom) PCRamplificationof cDNA
corre-spondingtothe DMB gene; the arrowindicatesthe expected DMB
productof 320bp.Namesofcell lines in each lane areindicatedabove bothpanels.Twoindependently fused SJOx .174hybrids were ana-lyzed. The higher molecularweightband present in some samples
(indi-catedbythearrowheads)islikelytohavebeenamplifiedfrom genomic DNAcontaminatingthe cDNApreparationssince bands of the same sizeareamplifieddirectly from genomicDNA(lanemarked "Gen.
Vavy"). ,
Priess BLS-1.DR4 6.1.6.DR4 SJO.DR4
AntigenPresenting Cell
Figure 2. DR4w4-transfectedBLS cellsareunabletomediate class II-restricted presentationof native exogenousprotein antigenstoT cells.
(A)Lymphokine production byaTr-specificT cellhybridomain re-sponsetoDR4w4-expressingAPCs incubated with 0.4,kMofatryptic digest of TT peptides (solid bars)ornativeTT (hatched bars). (B) Lymphokine production byaninfluenzaHA-specificTcellhybridoma
inresponsetoDR4w4-expressingAPCs incubated with 1.0 AMHA
307-319 peptide (solid bars)or50
ttg/ml
fixed influenza virus(hatched bars).Dataarepresentedasproliferationof theIL-2/IL-4-dependentcellline, HT-2,inresponsetolymphokines produced bythe T cell
hybridomas.The dataaremeanvalues of[3H] thymidine incorporation fromduplicate culturesinrepresentative experiments;cpmvalues
re-sultingfromresponseof TcellstoAPCs in the absence ofantigenhave beensubtracted.
cellswasefficient. Theinabilityof themutantAPC T2.DR4w4 cellstopresenteither nativeTTorinfluenzaHA tothe
DR4w4-restricted T cellswascomparableto the BLS cells (Fig. 2).
BLS cells lack HLA-DM mRNA. The defects in class II-mediatedantigen presentation inBLS cellsaresimilartothose observed in the invitro-generatedmutantcell lines 9.5.3(28),
2.2.93 (11), 721.174 (13), and T2 (14). Thesemutants each lackorhave mutations in MHC-linked genesrequiredfor
pre-sentation of native class II-restricted antigens, recently identi-fied as the DMA and DMB genes (10, 11). The DM genes
mapin the classIIregionof theMHC and contain 5'regulatory
sequences thatare similar tothe promoter regionsof the DR, DQ, and DP structural genes (12). Because the mutations in
BLS cells areintrans-acting factors requiredfortranscription
ofthe classIIstructuralgenes,wehypothesizedthatBLS cells would also be deficient inexpressionofDMgenes. Indeed, in
a previous study, the SJO cell line from BLS group IV had
been examined for DMmRNA,withevidence suggesting these
cellsare unabletoproduceboth DMA and DMB (32).
APCR assay was used to detect DMA and DMB mRNA
in cells representative of the four BLScomplementationgroups, BLS-1, BLS-2, 6.1.6,and SJO(Fig. 3). The amountof PCR-amplified signal correspondingtotheexpectedDMA and DMB
productsin each BLS cellwascomparedtothat ofawild-type
B cell line Vavy and the DM-deletion mutant T2. All four
BLS cells weredeficient in DMB mRNA (Fig. 3, bottom). In
contrast, assays ofBLS-1, BLS-2, and 6.1.6 showed a weak
band corresponding to DMA mRNA, while SJO lacked this band(Fig. 3, top). Theidentity of theputativeDMA band in
BLS-1 cells was confirmed by DNA sequencing ofthe PCR product. The difference in intensity of DMA bands between
wild-typeB cells and these BLS cells suggeststhat BLS cells contain reduced amounts of steady-state DMA mRNA. This observation is consistent with thereportthat BLS-1 cells also containreduced but detectableamountsofDQA1 mRNA(17).
These data indicate that thetrans-actingfactorsmissingin each of the four BLS cells, representingall fourBLS
complementa-tion groups, arerequired for transcriptional activation of both
the DMA and DMBgenes, althoughDMAexpressionis
appar-ently regulated byadditionalparameters.
Fusion ofBLS cells with the HLA class HI deletion mutant 721.1 74restoresawild-typeAPCphenotype.Cell fusion studies
have been usedtodemonstrate that the defects in class IIgene transcription in BLS cells arelinked to the absence of
trans-actingfactors encoded outside of the MHC (6, 7). To
investi-gateifafunctional APC phenotype could also be restoredby trans-actingfactors,wefused BLS-1(DR3,5)orSJO(DR5,7)
cells with 721.174. The latter line contains a deletion in the class II region of the MHC thatremovesboth structural class
Table 1. Fusion of BLS Cells with Mutant .174 Restores Expressionof DR Epitopes
mAb recognition of DR epitopes (meanchannelfluorescence) Cell line All DR(L243) DR3(16.23)
.174 8 10
BLS-1 12 15
BLS-1.DR3 680 84
BLS-1 x .174Fl 818 460
Mat 2996 734
SJO.DR4,6 13 nd
SJO x .174F3A 1558 nd
Anti-DR mAbbindingtolivecells was detectedby flowcytometry. Matisawild-typeDR3 B-LCL.BLS-1 contains anendogenous DR3
genethat is expresseduponfusionwith .174.SJO.DR4/3containsonly
aDRB1*0401 transgene.nd, notdetermined.
presentation of exogenous protein antigens, but is presumably intact for thetrans-acting regulatory genes defective in the BLS lines. Fusedhybrid cells BLS-1 x .174 and SJO x .174 were isolated and assessed for expression of endogenous class H
molecules in the BLS cells. The hybrid cellsexpressedabundant amounts of endogenous DR molecules detectable with mAb L243 that recognizes mature DR heterodimers (Table I) and by mAb SFR3-DR5 (33) thatrecognizesDR5 molecules (data
notshown). The SJO cells also containedatransgeneencoding
DR4w4p
chains;however, surfaceexpressionof DR4w4 mole-cules was only observed aftertranscriptional activation ofen-dogenousDRalpha chainexpressionin the hybrid cells(Table
I and datanotshown).
The DR af3 structure in these hybrid cells, as well as the previously characterized BLS-1 x SJOhybrid cells (8), was
investigatedusingthe assay for classIIdimerstabilitydescribed above. Upon fusion of BLS-1 orSJO cells with each otheror
with 721.174, the amount of SDS-stable dimercorresponding
totheendogenousDR geneproductswaspresent inwild-type
amounts(Fig.1, top). Thestructureofendogenouslyexpressed DR3molecules in the BLS-1 X.174 fused cellswasalso investi-gated using the conformationally-sensitive anti-DR3 mAb 16.23. Fusion of BLS-1 with .174 resulted in production of DR3 moleculescontainingthemAb 16.23epitope;theamount
of mAb 16.23 bound wassignificantly greater than that bound
to BLS-1.DR3 transferents and comparable to the wild-type
DR3Bcell line Mat(TableI).Identicalresultswerepreviously
obtained afterfusion of BLS-1 and SJO (8). Taken together,
thesedata indicate that fusion with 721.174 results in the pro-duction of DR molecules that have the sameconserved
struc-tural features asDR molecules present in wild-typeB cells, a
phenotype that ismarkedlydistinct fromDRmolecules in BLS and mutant APC such as 721.174. These results are simply explained if the trans-acting factors that mediate classH
struc-tural gene transcriptionalsomediatetranscriptionof genes
re-quiredfor afunctional APCphenotype.
Presentation of exogenous native antigenwasalsorestored
in SJO x .174 hybrid cells (Fig. 4). In marked contrast to
SJO.DR4w4 cells, the SJO X .174 cells were able to present nativeTT(Fig.4A)ornative HA(Fig.4B)totheappropriate DR4w4-restricted murineTcellhybridomas. While the presen-tationofHApeptides,eitherpreprocessedorderived after
cellu-Am.
o 100 x E
E.
30o 10
S2 10
0
33-
I--
0.3-300
U100 x
E 3
a. 30
-c
° 10*
2 3.
IL 1.
I-0.3-L_
- --_
Prlm SJO.DR4
AntigenPresenting Cell
Prim SJO.DR4 Antigen Presenting Cell
Figure4. Fusion ofSJO cells withtheHLAclassIIdeletionmutant 721.174 restores theability of SJOto present native exogenousantigens
to Tcells.(A)Lymphokineproduction byaTr-specificTcell
hybrid-omain response to DR4w4 B cell linePriess, SJO.DR4w4orhybrid SJO x .174cells incubated with0.4 sMTF peptides(solidbars) or
0.8
AM
nativeTf (hatched bars). (B) Lymphokine production by aninfluenzaHA-specificTcellhybridoma in response toPriess,
SJO.DR4w4 orSJO X .174 cellsincubatedwith 1.01MHA307-319 (solidbars) or 50pg/mlfixed influenza virus (hatched bars). Data are presentedasproliferation oftheIL-2/1L4-dependent cell line, HT-2.
Data are meanvalues of[3H]thymidineincorporation fromduplicate culturesinrepresentativeexperiments.
larprocessing of influenza virus,wascomparable inPriess and SJO x .174 cells, presentation of TT (native or
trypsin-di-gested)wasreduced inthe SJOx .174cells, relativeto
wild-typeB cells, inmultiple experiments.
The nearly wild-type APC phenotype of the hybrid cells promptedus tocheck thehybrid cells for the amount of expres-sionof HLA-DMA and -DMB, the MHC-linked genes required forfunctionalantigenprocessing.Fusion of SJO with 721.174 resulted in the presence ofPCR-amplifiedproducts correspond-ing totheDMA andDMB genes(Fig. 3). Since 721.174 has
ahomozygousdeletionof the MHC classHregion that includes the DMA and DMB loci, these data are consistent with the explanation thatthetrans-actingfactor present in 721.174 that
restoresclassHgenetranscriptioninSJO alsorestores
transcrip-tionof theDMAandDMB genes.
Discussion
An HLA class H null phenotype typifies each of the type II
defects manifest as thisimmunodeficiency. Restoration of class II expression in BLS lines by transfection of fully competent exogenous class II genes restores the ability of these cells to presentantigenic peptides to T lymphocytes. Notably, however, class II-transfected BLS cells remain incompetent to present protein antigens to the same Tlymphocytes, indicating a
persis-tent deficit in antigen processing and presentation. We have evaluated B cell lines from different BLS complementation groups tocharacterize thisantigen presentationphenotype, and found that all transfected BLS cells tested present preprocessed
peptidesto Tlymphocytes, but donotprocess and present epi-topes derived from whole proteins. Genetic complementation of the BLS defect by fusion with acell lacking genes in the class IIregion of the MHC restored both classIIexpression and
afully competent APC phenotype. Thus, each of the molecular defects that silences classIIstructural geneexpression in BLS also results in a defective APC phenotype, providing strong evidence for coregulation of these pathways.
Thedefective APC phenotype in BLS has a structural corre-late, as demonstrated by analyses of class II
af3
dimer stability and conformation-dependent anti-DR3 mAb epitopes. After roomtemperature incubation in lowconcentrations of detergent, DRmolecules in all BLS transferent lines tested showed greatly reducedstabilityascompared to class II molecules inwild-type B cells; fusion of the DR-transfected BLS cells with other class II negative mutants again restored a wild-type phenotype, in this case the stability of the class IIa/3 dimer. Recent studies ofDM-deficient mutant T2 and 9.5.3 cells expressing the murine class IImolecules, I-AdorI-Ak,have shown thatI-Aa/3dimer stability in SDS maynotbe strictly correlated with the abilitytomediatepresentation of native exogenous antigens(34, 35).
I-Ad molecules, but not I-Ak molecules (34, 35), exhibited markedSDS-instabilityyet retained thecapacitytopresent
cer-tain epitopes derived from exogenous protein antigens. Con-versely, SDS-stable I-Ak molecules were unable to mediate presentation of other epitopes (35). This differential behavior of the murine I-A molecules in the mutant human cell lines may be due to differences in avidity of these I-A alleles for the invariant chain-derived CLIP peptides. Although all DR
molecules showed greatly reducedSDS-stability in the BLS-1 cellline,weobserved subtle allelic differences inDRaf3 stabil-ity that may correlate with the differential avidityof these DR
alleles for the CLIPpeptides (31) and, therefore, may reflect
allele-specificformationof selected stablepeptide/classII
com-plexes. However, in deficient APC containing human class II
alleles, SDS-unstable class H af3 dimers are universally ob-served.
The genetic defects in three of the BLS complementation
groupsareknown; each involves defectivetranscriptionfactors requiredforcoordinate expressionof HLA class II genes.The
transcription factor defective in cells of complementation
groupsI and IV, suchas BLS-1 and SJO, is apparently RFX, sincepurifiedRFXprotein complementsDRAtranscription in these cells(36).An RFX heterodimer mostlikelyinteractswith
consensus X box elements present in HLA class II upstream regulatory regions (36). A deficiency in an X box binding protein in the 6.1.6 cell line, complementation group III,has alsobeen reported (37). Thedefective APC phenotype mani-fested in each of these three distinct complementation groups suggests that genesrequired forantigen presentationare coregu-latedbythesamesetof transactivators asthe class IIstructural
genes. Cells in BLS complementation group II, such as
BLS-2, aredefective in the CIITA protein (38),aprotein thatmay
notdirectly bind DNA (38) but which likelyactsinboth consti-tutive andy-interferon-stimulated class II transcriptional activa-tion (39, 40). Since BLS-2 cells lackDMB mRNA,CIITA is likelytobe involvedinregulation of genes required for antigen presentation (seebelow), suggesting that these cells would dis-playa defective APC phenotype upon transfection of class II genes. Indeed, a recent report(41 ) shows that transfection of the CHTA gene into BLS-2 cells results in endogenous DR andDMgeneexpression, indicating coordinate transcriptional regulation of these diverse class II genes.
Since thedefective APCphenotypein BLS cells is function-ally similar tothe phenotype of previously described in
vitro-generatedcellmutantsdefective in the HLA-DMA and -DMB genes, weevaluatedmRNAlevels for DMA andDMBin each ofthe four BLScomplementationgroups. mRNA forDMBwas indeed absent in all BLS cells tested, andwasrestored by
trans-actingfactors upon fusion withaDM-deficient cellline,
coordi-nate with class II expression and APC function. Amounts of
DMAmRNA, in contrast,werelowbut variable in the distinct BLS cell lines tested and also apparently increased in the SJO x .174 hybrid cells. This discrepancy between HLA class II A and B gene regulation may be a general phenomenon, since
expressionof both theDQA1 (17) and DQA2 (42) genes have also been previously described in cells lacking expression of
DQB. Thus, these data suggest that the absence of functional HLA-DMproteins in the BLS cells may account for the defec-tive APC phenotype.
Interestingly, the upstreamDNAsequence proximal to the firstexonof theDMAandDMBgenes containsHLAclass
II-likeconsensus sequenceelements, theX andYboxes, shared with class IIDR, DQ, andDP genes (12); and like the class IIstructural and invariant chain genes, theDMgenes arelikely
tobecoordinately upregulatedin responseto y-IFN (43). The presence of these similarregulatoryelements supports the
inter-pretation that each of thetranscriptionfactors defective in the BLScomplementationgroups alsoact onthe promoter elements for the DMA andDMB genes, and in this way, function as a
coordinateregulatorof the separate pathways of structural class II geneexpression andfunctional antigen processing and pre-sentation. However, thetranscriptionfactors absent in the BLS cells may be involved inregulationof additional genes required foroptimal antigenprocessing andpresentation. Accordingly,
wehave observedadecrease inamountsofintracellular
invari-ant chain in each of the four BLS cells described here (S.
Kovats and J. S. Blum, unpublished observations),consistent with coregulation of invariant chain gene transcription via 5'
XandYboxconsensus elements(16).
The conservation of shared transcriptional regulatory
ele-mentsprovidesasimpleandeffective mechanismfor coregula-tion ofthese twodistinct pathways, and is thus an interesting
geneticmodel forthecoevolution of distinct geneswhose
prod-uctsparticipate in different aspects ofa commonintracellular process.Asthe class II
a/3
dimerstabilityexperiments demon-strate, products of genes involved in APC function appear todirectly interface with the products of the class II structural genes themselves to produce fully competent APC.
Further-more,both the structural class IIpathwayand theantigen
pro-cessing/presentation pathway likely contribute to the clinical
immunodeficiencyassociated with BLS,implyingthattherapies directed at the coordinate regulatory elements rather than the
We thank P. Whiteley and D. Zaller for T cell hybridomas, R. DeMars and R.Greenwood for721.174cells, andJ. Leefor BLS-1 andBLS-2
cells.We also thank ourcolleagues for helpful discussionsand H. Chase for manuscript preparation.
This study was supported by National Institutes of Health grants
(AI-33418 andAI-31241) andtheArthritis Foundation(J. S. Blum).
S. Kovats wassupported byaNationalResearchServiceAwardfrom theNational Institutes ofHealth.
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