Defective major histocompatibility complex class I expression on lymphoid cells in autoimmunity

Defective major histocompatibility complex

class I expression on lymphoid cells in

autoimmunity.

Y Fu, … , X Li, D L Faustman

J Clin Invest.

1993;

91(5)

:2301-2307.

https://doi.org/10.1172/JCI116459

.

Lymphocytes from patients with insulin-dependent diabetes mellitus (IDDM), a chronic

autoimmune disease, have recently been shown to have decreased surface expression of

MHC class I antigens. Since IDDM and other autoimmune diseases share a strong genetic

association with MHC class II genes, which may in turn be linked to genes that affect MHC

class I expression, we studied other autoimmune diseases to determine whether MHC class

I expression is abnormal. Fresh PBLs were isolated from patients with IDDM, Hashimoto's

thyroiditis, Graves' disease, systemic lupus erythematosis, rheumatoid arthritis, and

Sjogren's syndrome. Nondiabetic and non-insulin-dependent diabetes mellitus patients

served as controls. MHC class I expression was measured with a conformationally

dependent monoclonal antibody, W6/32. Freshly prepared PBLs from the autoimmune

diseases studied and the corresponding fresh EBV-transformed B cell lines had decreased

MHC class I expression compared with PBLs from normal volunteers and

non-insulin-dependent (nonautoimmune) diabetic patients. Only 3 of more than 180 donors without

IDDM or other clinically recognized autoimmune disease had persistently decreased MHC

class I expression; one patient was treated with immunosuppressive drugs, and subsequent

screening of the other two patients revealed high titers of autoantibodies, revealing clinically

occult autoimmunity. Patients with nonautoimmune inflammation (osteomyelitis or

tuberculosis) had normal MHC class I expression. Autoimmune diseases are characterized

by decreased expression of MHC class I on lymphocytes. MHC class I […]

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Defective

Major Histocompatibility Complex Class I

Expression

on

Lymphoid Cells in Autoimmunity

Yineng Fu, David M. Nathan, Fangqin Li, Xiangping Li, and Denise L. Faustman

Immunobiology Laboratories of the Diabetes UnitandMedical Services, Massachusetts GeneralHospital and theDepartmentofMedicine, Harvard MedicalSchool, Boston,Massachusetts02114

Introduction

Lymphocytes from patients with insulin-dependent diabetes mellitus (IDDM), a chronic autoimmune disease, have re-cently been shown to have decreased surface expression of

MHC class I antigens. Since IDDM and other autoimmune

diseases share a stronggenetic association with MHCclass II genes, which may in turn be linked to genes that affect MHC class I expression, westudied other autoimmune diseases to

determinewhether MHC class Iexpressionis abnormal. Fresh

PBLs wereisolated from patients with IDDM, Hashimoto's thyroiditis, Graves' disease, systemic lupus erythematosis,

rheumatoidarthritis,andSjogren's syndrome. Nondiabeticand

non-insulin-dependent diabetes mellitus patients served as

controls.MHCclass Iexpressionwasmeasured witha

confor-mationally dependent monoclonalantibody, W6/32. Freshly

prepared PBLsfromtheautoimmune diseases studiedand the

corresponding fresh EBV-transformed B cell lines had

de-creased MHC class I expression compared with PBLs from normal volunteers and non-insulin-dependent

(nonautoim-mune)diabetic patients.Only3 of more than180donors

with-out IDDM or otherclinically recognizedautoimmunedisease

had persistently decreased MHC class I expression; one

pa-tient was treated with immunosuppressive drugs, and subse-quentscreening ofthe other twopatientsrevealedhigh titers of

autoantibodies, revealing clinically occult autoimmunity.

Pa-tients with nonautoimmune inflammation(osteomyelitisor

tu-berculosis)had normalMHCclass Iexpression. Autoimmune

diseases are characterized by decreased expression of MHC

class Ionlymphocytes. MHCclass I

expression

may be

neces-sary for self-tolerance, and abnormalities in suchexpression

may lead to autoimmunity. (J. Clin. Invest. 1993.

91:2301-2307.) Key words: major histocompatibilitycomplex-class I .

insulin-dependent diabetes mellitus *Graves' disease *

Hashi-moto's

thyroiditis*systemiclupuserythematosis * rheumatoid

arthritis

Address reprint requests to Dr. Denise Faustman, Immunobiology LabsoftheDiabetesUnit,MGHEast, Bldg. 149, 13thStreet,

Charles-town, MA02129.

Receivedforpublication 3 June 1992 and in revised form 21

Jan-utarv1993.

Insulin-dependentdiabetes mellitus (IDDM)'is characterized

by autoimmune destruction of the insulin-producingbeta cells

of the pancreas (1). Similar to other autoimmune diseases, IDDM has agenetic predisposition.Inthe absenceofan

identi-fied specific geneabnormality, the strongest genetic

associa-tionshavebeenbetween IDDM and genesencodingthe MHC

class IIpolypeptides (2-4). Similar linkagehasbeen shownfor

otherautoimmunediseases, including Graves' disease, Hashi-moto'sthyroiditis, systemiclupuserythematosis,and

autoim-mune adrenal insufficiency (5-7). MHC class II molecules

function to present foreign proteins to T lymphocytes. The

strong association between autoimmune disease and MHC

classII geneshassuggested that abnormalities in MHCclass II

geneproducts playacentralpathogeneticrole inautoimmune

diseases.

Ourrecentworkin IDDMhasshownthat in human(and

murine) autoimmune diabetes, antigen presentation and

ex-pression ofthe MHCclass I pathway is defective (8).

More-over,completeorpartial transgenic

disruption

of MHC classI

expression

alone is sufficient to induce a syndrome ofmild

autoimmunediabetes in micethat areotherwisenot

suscepti-ble todiabetes. Finally, preliminary evidencein a mouse model

ofspontaneousautoimmune diabetes (thenonobesediabetic

[NOD] mouse)linksthedefective expression ofMHC class I

moleculestogeneslocatedwithintheMHCclass IIregion (8);

these geneswithin the MHC classIIregion code for proteins

that controlendogenouspeptide delivery into the endoplasmic

reticulumand allow properMHC class I assembly and

subse-quentMHCclass I cellsurfaceexpression (8-16). Interruption ofthese MHC classII-linkedgenesuniformlyresultsin

low-eredMHC classI

expression

onlymphocytes.The central role

of MHCclass Iexpressionanddiseasecourse is wellillustrated

in theNODdiabeticmouse as well as inidentical twins discor-dantfor IDDM. Independentof genotype, only the subset of

NOD mice with decreased MHCclass I expression develops

diabetes. In

identical

twins discordant forIDDM,only

identi-caltwins withIDDMhavereduced MHCclass Iexpressionon

peripheralbloodlymphocytes;thenondiabetictwins have

nor-mal or nearnormalMHC class Iexpression.From this work, it appears that proper MHC class I presentationmay be a

path-wayfor self-toleranceand if altered,regardlessof genotype, can

lead toautoimmunediabetes.

1.Abbreviations

utsed

inthis paper: IDDM,insulin-dependent diabetes

mellitus;MAD, meanantigen density; NIDDM,

non-insulin-depen-dentdiabetes; NOD, nonobese diabetic.

Abstract

J.Clin.Invest.

©)

TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/93/05/2301/07 $2.00

Becauseofthestrongassociation of autoimmune diabetes

(IDDM) with theMHC class I defect, we investigatedother

autoimmune diseases fordefective MHC classIexpressionon

fresh peripheral blood as well as on freshly produced

EBV-transformedBcell lines.

Methods

Patients. Patients with IDDM (n = 54), Graves' disease (n = 36),

Hashimoto's thyroiditis (n=16), SLE (n= 14), and RA (n= 16) were identified from the out-patient clinics of the Massachusetts General Hospital. To be included,patients had to have histories and physical findings consistent with theirdiagnoses. Additionally, for Graves' dis-ease,Hashimoto'sthyroiditis,SLE, and RA, serologicalevidence ofthe specific autoimmune disease (long-acting thyroid stimulating, antithy-roid, and anti-DNA antibodies, and rheumatoid factor, respectively) wasrequired. Patients with IDDM haddiabetes onset before age 30, wereketosis prone, and were treated continuously with insulin. Nor-mal volunteers andpatients with non-insulin-dependent diabetes mel-litus (NIDDM) who had no personalhistory and no family history of any of the diseases listed above were included as control subjects. NIDDMpatients (n =42) developedtheirdiabetes after age 45 and had nohistory of urineketones.One NIDDMpatient was on high dose steroidsfor kidneytransplant. Five lupuspatients, one Sjcgren's

pa-tient,andtworheumatoid arthritispatientsweretreatedwith low dose steroids (generally less than 10 mg Prednisonedaily)for their underly-ing disease process. Sevenpatientswithnonautoimmune inflamma-tion(sixwith osteomyelitis and one with tuberculosis) were studied as anadditionalcontrol group.Fiveofthesix patients with osteomyelitis alsohad NIDDM.

Study design.Apreliminary studyon MHCclassI expressionin fresh peripheral blood lymphocytes from controls ranging in age from 12 to 45 yrrevealed no age, sex,orHLAhaplotype-related changes in this marker. MHC classIexpressionwasslightlyage related between ages 2 and12, therefore IDDMpatientsof this age groupwereanalyzed

with age-matched controls. Patients with the otherautoimmune

dis-eases wereall older than 12 yr.Acomparisonof the resultsofrepeated

assaysofMHC class Iexpressiononcontrolsperformedonthesame

dayconsistentlyrevealed< 1% absolutedifferences in percentage(95% confidence interval) and <2% absolute difference (95% confidence

interval) in mean antigen density between repeated analysesofthe

samesampleorbetweendifferentnormal individuals whowere

ana-lyzedforMHC class I antigensusingeither MHC class Iantibodies

(W6/32, BBM1) orantibody directed to

32-microglobulin,

thecell

surfaceprotein whichassociates ina 1:1fashionwith MHC class I.An

example of thehistograms ofMHC classIexpressionforeightnormal

donors,measured onthesameday,is shown in Fig. 1. In contrast, greatervariation was seen in the same normal sample analyzed on

different days duetothewell-describedanalysis dayeffect of flow

cy-tometry.Ananalysisofcovariance withtermsfor age, sex, group(same

controlsample compared with repetitive self-sample versusanother controlsample),HLA type, andanalysis day,revealedonlya

signifi-canteffect ofanalysis day (P=0.0004). The results of thispreliminary

study dictated that isolated peripheral blood lymphocytes from pa-tients and controlsubjectsbe obtained andanalyzedonthesameday. Patient and controlEBVlineswerealsoanalyzedonthesameday for directcomparison. This studydesignwasalsochosentoprevent the changes in lymphocyte surface markers thatoccurwithincubation,

freezing, and fixation ( 17, 18).Apaired t test was used to test whether lymphocytesurface markers (MHC class I)weredifferent between pa-tients ineachgroup and theirpaired controlsubjects.Dataarereported

asmean±standarddeviation unless noted otherwise.

Flowcytometer analysis. PBLs were prepared, as previously

de-scribed,from 40ccofbloodfreshlycollected inheparinizedtubes (17).

Inadditiontotheanalysis of PBLs, newpermanentBcelllineswere

established byEBVtransformation of freshBcellsfrom bothpatients

L-E

z

=:

.1 Meee

Log FluorescenceIntensity

Figure1.MHC classIexpressiononPBLsfrom eightnormaldonors

analyzedon thesameday.PBLswereanalyzedwith W6/32,a

monoclonalantibodytohuman class I.

andcontrols. Approximately2-3 moafterEBVtransformation,the newlytransformed patientcelllineswererandomlypaired with newly transformed EBV cell lines from control individuals and analyzed by

immunofluorescence.MostnewpatientEBVcelllineswereanalyzed

atleasttwice with differentsimultaneously pairednewcontrol EBV cell lines. Patient and control cell lines appeared stable during the time frameof this study;the data presented represent datafromonly oneof theanalysis days.Fluorescenceactivatedcell sorticanalysiswasusedto

quantitatecellsurface differencesin class Idensity. This techniquewas chosen becauseof theabilityof this methodtoquantitate accurately

epitopedensityoverfourlogsofimmunofluorescence.

For theanalysisof MHC classIdeterminantsonfresh cellsorEBV

lines,mouse monoclonalantibodyW6/32 (American Type Culture

Collection, Rockville, MD), which recognizes all alleles of human MHC class I,wasusedat afresh stock concentration of0.88,g/mlwith theaddition of 100 microliters for 1 X 106EBVcellsorfreshstockat

1.6

,gg/ml

with the addition of 100,gfor 1 x 106PBLs. This

concentra-tionofantibodyrepresentsan excessoverthe 50%saturationpointfor eachcell population but eliminates irrelevant Fc receptorbinding

whenusedatahigherconcentrationorlow-affinity bindingtothe class

Idimer, i.e.,classIand

,32-microglobulin

withoutpeptides.The

puri-fied W6/32 antibody wasused with goat anti-mouse IgG2a FITC (Tago,Burlingame, CA)for indirect immunofluorescenceat a

concen-2302 Y. Fu,D. M.Nathan, F.Li,X. Li,andD. L.Faustman

I I I

---TableI.Expression ofMHC ClassIDeterminantsonFreshly IsolatedLymphocytes froma Variety ofAutoimmuneDiseases

No.of

paired Expression of MHC samples class Iantigens*

% MAD

IDDMcontrol 54 82.4±13 6.0±4.5

90.9±12 9.7±6.7

P<0.001 P<0.O01

NIDDMcontrol 42 97.4±2.7 15.5±7.6

96.6±3.4 16.3±8.4

P=0.20 P=0.37

Hashimoto's control 16 77.7±21 6.9±5.6

94.5±3.1 10.0±7.7

P=0.003 P=0.001

Graves' control 36 79.5±22 7.4±4.5

91.4±14 12.3±7.2

P<0.001 P<0.001

Lupus control 14 81.2±20 7.4±4.5

95.4±2.5 14.4±11.4

P=0.01 P=0.009

Rheumatoidarthritic

control 17 80.4±18 10.0±11.5

93.8±7.7 13.1±11.6

P=0.01 P=0.02

* Values are

mean±standard

deviation forapairedt testbetweena

patientandsimultaneouslypairedcontrolsamples.Patients less than 12 yrold also hadanage-matchedpaired sampleduetotheslight differences in MHC classIexpressionin younger children.

tration greater than 100-foldexcessofthe50%saturationpoint.The W6/32 antibodywasselected because it isconformationally

depen-dent and thus demonstratesmostdramaticallythelowered MHC class Ionautoimmune cells (19). Subsetanalysis offreshperipheralblood

lymphocyteswasperformedby two-coloranalysis using directly

FITC-conjugated W6/32 antibodywithdirectlyphycoerythrin (PE)-conju-gatedBi antibody(B cellmarker), Mo2antibody(macrophageand monocytemarker), orCD2antibody(T cell marker) (Coulter Elec-tronics Inc.,Hialeah, FL).Fortwo-coloranalysis, FITCemissionwas separated from PE emission using appropriate filters, differentially conjugatedbeads, aswellas acontrolsamplelabeledwith CD4 and

CD8for thedeterminationoftheappropriate compensation.All pheno-typingsampleswere analyzedon a flow cytometer(Elite; EpicInc., New York); more than 5,000 cells persamplewere analyzed. The

sampleflowrate wasmaintainedatmorethan350cells/second.The

operatorwasmaskedtosample identity. For PBL, a gate on theFACS® machine (Becton Dickinson & Co., Mountain View, CA) was set to exclude small nonlymphocyte debris using a T200 antibody (KC57; Coulter Immunology) specific foralllymphocyte subpopulations. In addition, viable cells were identified by forward light scatter intensity andexclusion of propidium iodine which labels dead cells. For EBV analysis,agatewasset toexclude debris and includedEBVcellsof the largesize on log fluorescence. EBV cells before analysis were in log phase of growth, never frozen and determined to have viability of

>95%. The percentage of positive cells was determined using a three log decade histogram with a 95% region for isotype control. Back-ground immunofluorescence, determined by an isotype-matched, FITC-conjugated, or PE-conjugated antibody was virtually identical in allcasesand always less than 5%.

Due to thedifference in mean antigen density (MAD) of MHC classI onmonocytes versuslymphocytes, two gates were set to analyze unseparated PBLs with single-color immunofluorescence using the W6 / 32antibody. For data calculations in TablesIand II, a gate was set

on the small-sized but abundant nonmonocyte population. As ex-pected, identical statistical trends were observed for the data in Tables I andIIwith a gate set only on the very large-sized but less abundant monocytesubpopulation (data not shown). For the two-color immu-nofluorescence presented in Table III, an open gate excluding only nonlymphocyte debris was utilized. This open gate was used to deter-mine thedensity ofMHC class I on specific lymphocyte

subpopula-tions, i.e.,Tcells, B cells, and monocytes.

Results

As previously reported, MHC class I antigen

expression

on

fresh peripheral blood lymphocytes from patientswith IDDM

was consistently depressed (Table I, Fig. 2). Lymphocytes

fromNIDDMpatients,on theotherhand, hadasimilarmean percentageof MHC classIdeterminantscompared with

nondi-abetic controls (97.4±2.7vs.96.6±3.4,P=_0.20)

(Table I)

as

expected foradiseasewithout autoimmune

etiology.

In

addi-tion,

fresh lymphocytes from NIDDMpatients hada similar

density

of MHC classI

antigens

compared with fresh control

cells( 15.5±7.6vs. 16.3±8.4,P= 0.37). Interestingly, 3 ofthe

42 NIDDMpatients analyzedappeared to havesignificant de-creasesin MHC classI percentages or mean

antigen

densities

compared with

simultaneously

analyzed control samples. As

outlined in Methods, normal control samples from different

donorsanalyzedonthesameday demonstratedlessthan±1%

variability between individuals in percentage and/or less

than±2%

variability

inmean antigen density of MHC classI

antigens.

If the absolute

difference

inpercentageofMAD

be-TableII.Modification ofNIDDM Lymphocyte MHC Class I Expression by Underlying Autoimmunity or Steroids

NIDDMPatient 1* Control NIDDMPatient2t Control NIDDMPatient 30 Control

FreshPBL

% 93% 99% 78% 93% 65% 94%

MAD"I 7.08 17.7 7.2 13.5 9.3 18.2

EBVcell line

% 97% 96% 42% 82% 97% 99%

MAD 5.6 4.7 1.39 4.50 13.7 19.8

* NIDDM patient on chronic high dose steroids secondary to a kidney transplant. *

NIDDM

_{patientsubsequently found to have anti-smooth}

TableIII.MHCClassIExpression onSubsets ofPeripheral Blood Lymphocytesfrom VariousAutoimmuneDiseases

Macrophage/monocyte* Bcells* Tcells*

% MAD % MAD % MAD

A. Individualpatients

IDDM

patientl

control 97 7.8 56 4.1 91 5.6

99 26 94 14 97 7.1

Graves'patientcontrols 95 26 92 20 99 19

100 32 100 27 100, 21

Hypothyroidpatient control 96 18 96 28 99 13

99 41 99 35 99 20

Lupuspatient control 90 33 84 28 90 14

96 39 86 31 92 25

Sjogren'spatientcontrol 97 19 96 24 98 18

99 56 99 41 100 30

B. Combined autoimmunepatientst

MAD MAD MAD

Autoimmunepatientsn=53

paired samples 25±2.9 18±2.1 12±1.0

Controls 32±2.7 24±1.9 17±1.2

P=0.000 P= 0.000 P= 0.000

* Asdescribedin Methods, two-colorimmunofluorescencewasperformed,using directly conjugated W6/32 (MHCclass I monoclonal marker) with Mo2 (macrophage and monocyte monoclonal marker), B 1 (B cell monoclonal marker), orCD2(Tcell monoclonal marker) to quantitate therelative amountsofMHC class Iepitopesondifferentlymphocytesubsets. *_{Thesedata represent pooled valuesfrom 10lupuspatients, 15}

Graves' diseasepatients,8 rheumatoid arthritispatients, 10Hashimoto's patients,and 10 typeIdiabetics compared with theirpairedcontrols. §Asdiscussed inMethods, themeanantigendensityof MHC class Ionlymphocytesdoesnotvary betweenadults age 12-45 yr old. In contrast, MHC class Ionlymphocytes fromchildren, ages 2-12 yrold, isagedependent. ManyofourIDDMpatientswerechildrenand were agematched,therefore accountingfor theslightlylowermeanvaluesfor thedensity ofMHC class Ionthepatientsand matched controls in thisautoimmunecategoryof patients.

1l

Becauseofthelog fluorescencescaleformeanantigendensityandthefactthat the total numberofclass Imolecules is lessonTcellscomparedwith B cellsormacrophages,themagnitude ofclassIchangewasusuallyleastdramative in the T cell subset.Thisis well demonstrated in the above table for the Graves'disease patientwhere the T cellfluorescence isdecreased in thepatientbut the actual valuesareclosetothe control value.

tweenthepatient and control exceeded 1%or2%

respectively,

thepatientwasconsideredtohave abnormal MHC class I

ex-pression. ThreeNIDDMpatients demonstratedanunexpected

decrease(6%, 15%, and29%)inpercentageanddecreasedin

mean

antigen density

(

11%, 6.3%,

and

9%)

of MHC classI on

I.

A

I

.see

.

lymphocytes compared with their nondiabetic controls.

Fur-ther studieswereperformed in these NIDDM patients. One of thethree NIDDM patients was treated with high dose glucocor-ticoidsas well asimmunosuppressive drugs (CyclosporinA;

Sandoz, Basel, Switzerland; and Aziothioprine; Burroughs

C

L

1...I

m.

II"= I..610

*.I.m-,.P-*|H

loll-Fluorescence Intensity

Figure 2. MHC class IexpressiononPBLs fromautoimmune patients (dashed line) compared with control individuals(solid line). (A),IDDM; (B), rheumatoidarthritis; (C), lupuserythematosis; (D), Graves'; (E),

noninsulin dependent diabetes (NIDDM). Patient and paired controlwereanalyzedon

thesameday (see Methods).

C-0

L₃

D

2304 Y.Fu,D. M.Nathan,F.Li,X.Li, andD.L.Faustman

B

I

1 le lee low

f-l.

Wellcome,ResearchTriangle, NC)for akidneytransplant

(Ta-ble II, patient 1).Steroids andimmunosuppressive drugshave

knownlymphocyte-modifying effects and therefore might

mod-ify MHC class I expression on fresh peripheral blood lympho-cytes(20). Newly producedEBVtransformed B cells from this

patient demonstratedthe returnof normal MHC class I

deter-minantsinculture, demonstrating that the in vivodepression

in MHC class I was mostlikelysecondary to thein vivodrug effects (Table II). The other two NIDDM patients with low MHC class Iexpressiononfreshlymphocytes were not treated

withanymedicationthat could adversely affectMHCclass I

determinantsandhad no clinical orfamily historyof

autoim-munediseases.Alaboratory screenfor clinically silent

autoim-munityrevealedhigh titers of anti-smooth-muscleantibodies

inonepatientandanti-thyroid-colloid antibodies,

antimicro-somal antibodies, and antinuclear antibodies in the other

pa-tient, suggesting underlying autoimmunitywaspresent inthese

two NIDDM patientsidentifiedbylow MHC class I antigen

expression

on fresh peripheral blood lymphocytes. Newly

es-tablished EBV cell lines from both NIDDM patients

main-tained decreased MHCclass Iexpression with repeated

pheno-typing,

as

predicted

for agenetically controlled autoimmune

abnormality (Table II). Thesevenpatients with osteomyelitis

or tuberculosis had normal MHC class I expression with

99.3±0.59%

expression

and 13.5±3.3MADand thus were

vir-tuallyidenticaltothecontrolsamples performedonthe same

dayasexpected for a nonautoimmune butinflammatory

dis-ease.

Westudied fourgroupsof patients withotherautoimmune

diseases. The lymphocytes from 29 of 36 Graves' disease

pa-tients hadadecreased percentage ofMHCclass Ipositive

lym-phocytes withameanpercentageof 79.5±1.22 compared with

91.4±14percentpositive fresh lymphocytes forthe controls (P

< 0.001) (Table I). Decreased expression was similarly

re-flectedintheMADof MHCclass IepitopesonGraves'disease

lymphocytes compared with control lymphoidcells (7.4±45 vs.

12.3+7.2,

P <0.001 ) (TableI).

The fresh lymphocytes from 12 of14 lupus patientsalso

hadadecreased percentageof MHC class Ipositive

lympho-cytes compared with control lymphocytes (81.2±20 vs.

95.4±2.5;P= 0.01) (TableI); fresh lymphocytes from lupus

patients also hada depression in the MHCclass I density of

MHC classIepitopes(7.4±4.5 vs. 14.4±11.4) in 12of14

indi-viduals compared with simultaneously analyzed controls (P

=0.009).

15 of 16 patients with Hashimoto's thyroiditis had fresh

lymphocyteswith decreased MHC class I determinants

com-pared with thelymphocytes frommatched controls(77.7±21

vs. 94.5+3.

1,

P= 0.003); the mean density of MHC class I

antigens

wassimilarlydepressedinthesehypothyroid

individ-uals(6.9±5.6 vs.

10.0+7.7,

P=0.001)(TableI).

Thefresh lymphocytes from 16 of17rheumatoidarthritis

patients demonstrated a statistically significantdepression in

MHC class I expression for both the percentage of positive lymphocytes (P =

0.01

) as well as mean antigen density (P

=0.02) (Table

I).

Peripheralblood lymphocytes represent a diverse variety of

cell types, i.e., macrophages/monocytes, B cells, and T cells. These cells can be nonspecifically identified by flow cytometry

analyses bytheirdistinctsizes (T and B cells vs. monocytes), as

well as individually studied by two-color flow analysis with

cell-specific

antibodies incombination withaMHC classI

an-tibody. Both of these studies wereperformed on freshblood

samples in orderto determine if the defective MHC class I

expression in freshperipheral blood was confined to agiven

subpopulation of cells or expressed equally by all lymphoid

cells.In eachsamplefrom patientswith the autoimmune

dis-eases, differential gatingbycell size revealedsimilar,

statisti-cally significant,MHC class Idepression in all cellpopulations

compared with similarly gated fresh control cells. Follow-up

two-colorimmunofluorescence, which findsspecific

lympho-cyte cellular subsets, identified low MHC class I expression

proportionallyon Tcells,Bcellsaswellasmacrophages/

mono-cytesfromtype Idiabetics, Graves', Hashimoto's/

hypothyroid-ism,lupus,andSjogren's syndrome.Table IIIshows theresults

of this two-coloranalysisfor five different diseasesaswellasfor

allautoimmunepatients combinedand demonstrates the

non-exclusivenatureof the MHC classIdefectin different

lympho-cyte

subpopulation

fromfreshblood

samples

of

patients

with

diverse autoimmune diseases. Examples of individual

histo-gramsof the cellular subsetsareshowninFig.3. The

autoim-munehistograms for each cellular subset,

i.e.,

Tcells (A,B),B

cells (C, D) and macrophages (E, F) clearly demonstrate a

shift to the left, even when the y-axisfluorescence is

repre-sentedon alog scale.Because the total number of class I

mole-culesis lesson Tcells thanBcellsor

macrophages,

the

magni-tudeofthelog fluorescence changein class I

density

(fluores-A

II

I Il Uh4|1II,1HltI l*111ls_61111tees

. 1 1000

}/

I

I4

I-iWUUE I I1IIIUIUA 11Da1

.1 ieee

E

ix

I

II

:

_1-St..

m-rr

.1

B

IIaIw* 15", ,,,II 1,,-,I

.I IINS

D

rl

II

.1

F

_I

I

i08

. 1

Figure 3.MHC classIexpressionon T cells, B cells, and macrophages

fromtwotype Idiabetic patients (dashedlines;A, C, Erepresent

Patient 1 and B, D, F represent Patient 2) compared with matched control(solidline).All sampleswere analyzed on thesame day.

MHCclass Iexpression on T cells (A, B), B cells (C, D), and

mac-rophages(E, F)fromthe twodifferent type I diabetic patients are

shiftedtotheleftcomparedwiththe control.

Table IV. ExpressionofMHCClass I Determinants in EBVCellLinesfroma VarietyofAutoimmuneDiseases

No. of Expression of MHC paired samples class I antigens*

% MAD

IDDMcontrol 35 76.7±16.0 4.3±3.2

92.6±6.7 6.7±5.0

P<0.001 P<0.001

NIDDMcontrol 11 95.9±2.2 7.1±3.2

96.5±2.9 6.9±2.9

P=0.57 P=0.80

Graves'/Hashimoto's

control 9 84.6±18.9 4.7±2.9

96.3±5.3 7.7±5.3

P=0.03 P=0.02

Lupus control 7 59.1±31 2.7±1.4

88.0±8.8 6.3±3.2

P=0.02 P =0.004

Rheumatoidarthritis

control 15 75.9±11.7 3.8±1.5

90.5±7.0 7.8±2.8

P<0.001 P<0.001

* Values are

mean±standard

deviationforapairedttestbetweena

patientandsimultaneouslypairedcontrolsamples. Patients lessthan 12 yrold also hadanage-matched paired sampleduetotheslight differencesin MHC classIexpressionin younger children.

cence) was least

impressive

on T cells for all autoimmune

categories

of

patients.

Nonconformationally

dependent

polyclonal

anti-fl2-mi-croglobulin

serumwasusedon 10 IDDM fresh lymphocytes

compared with 10matched controls. Similartothetransporter

mutantcelllines suchasRMA-S,the

magnitude

ofthelowered

classI

fluorescence

was

significantly

lessdramatic. Themean

fluorescence for

f2-microglobulin

was 37.5±6.7 for IDDM

cells; themeanfluorescence for

32-microglobulin

was40.9±7.9

for matched control

(P

=0.

16). Presumably

becausea

confor-mationally dependent

antibody

suchasclassI

specific W6/32

requires both thepresence

of

classIaswellas

peptide

in the

groove of class I for

detection,

the diabetic cells have both

a

8

=0

A

rI I

\1

S

'1

I...,.~~~~~~~~~~~~~~~~~~~~~~~

X

'.k

L---IN

".,

empty unstable class I on the cell surface as well as less total class I on the cell surface, thus explaining why conformation-ally dependent antibodies dramatize the class I defect better in autoimmune cells as well as on mutant cell lines.

Asmentioned previously, many in vivo factors other than

genetics could be influencingMHC class I expression. The

pos-sible influence ofsteroids and/or immunosuppression in

modi-fying lymphocyte functions should be considered (20). To

controlfor thisvariable, newly transformedEBV B cellswere

produced from patients for each of the autoimmune diseases.

Theanalysis ofthese celllines eliminatedthe possibleinfluence

of concurrent medications or yet unknown serum factors. The percentage(76.7±16vs.92.6±6.7, P < .001 ) and mean

fluorescence intensity (4.3±3.2vs. 6.7±5.0, P < .001 ) of MHC

classIdeterminants is depressedin type IdiabeticEBVB-cell

linescomparedwithcontrol celllines(Table IV). Asexpected,

EBVcell lines from nonautoimmuneNIDDM patients didnot

show depressed numbers ofMHC class I cells (95.9±2.2 vs.

96.5±2.9, P= 0.57) or adifference incell surfacedensity of

MHC classIdeterminants(7.1±3.2 vs. 6.9±2.9, P=0.80). All

3 Graves' EBV cell lines, all 7 lupus EBV cell lines, all 15

rheumatoid arthritisEBVcelllines,and the cell lineestablished

from a patient with Sjogren's all showed a decrease in the per-centage(P=0.03,P=0.02,P=0.001, respectively)as well as

meanchannel fluorescence (P = 0.02, P=0.004, P <0.001,

respectively) of MHC classI lymphocyte epitopescompared

with simultaneouslyanalyzed EBV cell lines from controls

(Ta-ble IV). Fig. 4 shows the characteristic histograms obtained

from EBV cell lines analyzed with the monoclonal antibody

directedtoMHCclassIforagiven autoimmune disease

com-pared with

simultaneously

matchedcontrol EBV cellline.For

eachautoimmune disease, the leftward shift in MHC class I

fluorescenceintensity reflectsadecreased density ofMHCclass

Iepitopes in each transformedBcelllineandthus decreased

percentageof

positive

cells compared with controls.At notime

duringouranalysisofover80 new EBVcell lines from

autoim-mune

patients

didweobservemaintenance ofa normal MAD

of MHC classI determinants witha decreasedpercentage of

MHC class I

positive

cells; therefore, the existence ofatruly

negative MHCclass I Blymphocyte cell subpopulation is

un-likely.

Fig. 4demonstrates inthe samplehistograms from di-verseautoimmune diseasesthat themajor shiftin MADoften

111 10 100T 10 II

I1. I10 100 1000 .1I 1. 10 100 1000 .1 1. 1'0 100 1000

Figure4.MHC classIexpression on

EBV Bcelllines fromautoimmune

patients (solid line)compared with control individuals(dashed line).A, IDDM;B,rheumatoidarthritis;C, lupus erythematosis; D, Graves'; E, noninsulin dependent diabetes (NIDDM). Background

immunoflu-orescenceis shown andwassimilar for allthecelllines. Eachhistogram

pair representsasinglecharacteristic

patientand matched control.

2306 Y.Fu,D.M.Nathan,F. Li,X.Li,and D. L. Faustman

.1

.1 10 100 1000 .1 1. 10 100 1000

secondarilycauseshistogram overlap withbackground

fluores-cence and a resulting decrease in the percentage of positive

cells. Therefore, themeanfluorescenceaswellaspercentage of

positive

EBVcells for MHC class I aredecreased in

autoim-mune diseases. As expected EBV cell lines from

nonautoim-mune NIDDMpatientshad a virtuallyidenticalMHC class I

flow histogramtonondiabeticcontrols(Fig. 4 E).

Discussion

Analysis of fresh lymphocytes and newly produced

EBV-trans-formedBcell lines from Graves' disease, lupus erythematosis,

Hashimoto's

thyroiditis,

Sjogren's syndrome, and rheumatoid

arthritis

patients

revealed auniform decrease in MHC class I

expression

on

lymphoid

cellsnotobserved in control

individ-ualsorNIDDM,achronic nonautoimmune disease.Similar to

IDDM,acommonfinding inavariety of autoimmune diseases

withstronggenetic

association

with MHC classIIlinkage isa

paradoxical

defect

in MHC classI

expression

thatwaspresent

on alllymphocyte

subpopulations

andwasmostdramatically

observed witha

conformationally

dependent class I antibody.

Weidentifiedtwo NIDDM

patients

with decreased MHC class

I

expression

on fresh PBLand EBV celllines; follow-up

au-toantibody

screening

confirmed subclinical acute

autoimmu-nity.

In light of the recent identification ofnew genes in the

MHC class II

region

that

influence

MHC classI

expression by

supplying

endogenous

cytoplasmic peptides

toMHC class I in

the

endoplasmic

reticulum

(8-15),

it isinterestingtospeculate

that, similartoIDDM,a commontheme inautoimmunity is

defective

MHC class I

function (8).

Cell lines with induced

mutations in these MHC class

II-linked

geneshavedefective

MHC classI

expression

duetothe

faulty delivery

of

endoge-nous

peptides

associated with the abnormal MHC class II genes. This results in decreased classI

assembly

and surface

expression.

In the past, it has been difficult to

explain

how

defective

MHC class II could result in

autoreactivity

to

self,

since the known

function

of MHC classIIistopresent

phago-cytozedexogenous

antigens

to CD4 cells to elicit a

positive

immuneresponse. The

demonstration of

auniform decrease in

MHC classI

expression

of

lymphoid

cells in

autoimmune

dis-easesuggeststhat the

presentation

of endogenous

peptides

on

MHC classIisa

possible pathway

for self-tolerance. Similarto

transporter mutant cell

lines,

the defect in surface classI

ex-pressionwasmostdramatically observed witha

conformation-ally dependent

antibody,

thus

implying

thattotal surface classI

isnot onlydecreased in autoimmune

lymphocytes

but

presum-ably

peptide

emptyclassIis alsopresent onthe cell

surface

(21,

22). Interruption of this self-antigen

presentation

pathway, by

any

mechanism,

is sufficient for

autoreactivity

manifested

against

a

variety

oftarget organs

(8).

A

transgenic murine

model

with

decreased MHC classI

expression

alsodevelops

autoimmunity

(8).

Acentralunanswered question is howthe targettissueof

thevarious autoimmune diseases is specified.Perhaps the

tar-get ofanygiven autoimmune disease isdetermined by

non-MHG-linked genes determining target specificity.

Alterna-tively, interruption of specific peptide supply factor genes in

the MHCclass II region in combination with a specific MHC class Ihaplotypemaydeterminethe target tissue most vulnera-ble toautoimmuneattack. Further studies are required to

es-tablishthe

specific

mechanisms by whichMHC class Isurface

expression

and

presentation

are altered in autoimmune

dis-ease.

Acknowledgments

We are indebted to KathyHurxthal,R.N.,for her assistance in patient recruitmentforblood donations and LynneMurphy for her assistance

in thepreparation of the manuscript.

This work was supported by theMassachusetts General Hospital.

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