Staphylococcal exotoxin superantigens induce human immunodeficiency virus type 1 expression in naturally infected CD4+ T cells.

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Staphylococcal

Exotoxin

Superantigens

Induce

Human

Immunodeficiency

Virus

Type

1 Expression in

Naturally Infected CD4+

T

Cells

JAN E. BRINCHMANN,* GUSTAVGAUDERNACK, ERIKTHORSBY, ANDFRODEVARTDAL

Institute of Transplantation

Immunology,

The NationalHospital, 0027 Oslo1, Norway

Received13December 1991/Accepted 23 June 1992

Ahigh proportion ofStaphylococcusaureusstrainsof humanorigin produceone or moreexotoxins.Invivo,

thesetoxinsmaygiverisetoavarietyofclinicalsyndromes.Invitro, staphylococcalexotoxinshave beenshown

tobindbothtohumanleukocyte antigen (HLA)classIImoleculesonantigen-presentingcellsandtotheT-cell

receptorsonlargefractions of T cells. The result of this interactionmaybeproliferationofthe Tcells, T-cell

anergy,orapoptosis,dependingonseveralfactors, includingthe state of therespondingcellsand thepresence ofaccessorymolecules. Usingnaturallyinfectedperipheralblood mononuclear cellsdepletedofCD8+Tcells, we have shown thatstaphylococcal exotoxinsarepowerful inducers of humanimmunodeficiencyvirustype 1

expressionand thattheyinduceexpression atlowconcentrations and withgreaterefficiencythan otherT-cell

mitogens. Human immunodeficiency virus type 1 was produced entirely by CD4+ T cells in this model;

monocyteswereexpendablebothas a sourceofvirus andas a sourceof HLAclass IImoleculesaslongasother

cellsexpressingHLAclassIImoleculeswerepresent.The results suggest that infectionbyS.aureusmaybea

cofactorinthe

immunopathogenesis

ofAIDS.

During the asymptomatic phase of human

immunodefi-ciencyvirustype 1 (HIV-1) infection, fewer than 1 in5,000

CD4+ T cells are normally found to be harboring HIV-1 provirus(3, 20). Thesearemostlyrestingcellsandmustbe activated in order to produce infectious HIV-1. In vivo

activationofCD4+Tcellsisthoughttooccurpredominantly

in response to peptide fragments of antigen presented by

human leukocyte antigen (HLA) class II molecules (15).

However,thefrequencyof T cells which specifically

recog-nize a given peptide-HLA class II complex is only on the

orderof 1 in 10,000orless(1).Itfollows that the likelihood

that a particular antigenic peptide will activate an HIV-1

provirus-containing CD4+ Tcell issmall.

Staphylococci produceexotoxins(staphylococcal

exotox-ins [SE])which areamong themostpotentT-cellmitogens

known(16). TheyhavebeenshowntobindtoHLAclass II molecules outside the peptide-binding groove, and each exotoxin stimulates T cells bearing any of a number of

sequences encodedbytheT-cell receptor

VP

(16). Thus, in

contrast to antigenic peptides, each SE may stimulate as

manyas20% of all Tcells,apropertywhichhas earned them

the term "superantigens" (12, 16). Staphylococci are

fre-quentlyisolated fromHIV-1-infectedindividuals(14). More

than40%ofStaphylococcusaureus strainsproduce one or

moreexotoxins(8).Thus, SEsuperantigensmayfrequently

be available for activation of large subsets of T cells in

HIV-1-infected individuals. If abletoinduce HIV-1

replica-tion, such superantigenic T-cell activation might be an

important mechanism behind the ongoing production ofnew

HIV-1virionsinvivo(11).Inthis study,weshowed that SE

superantigensarepowerfulinducersofHIV-1 expressionin

naturally infected CD4+Tcells invitro. CD4+ T-cell

prolif-eration andthe expression ofHIV-1 were critically

depen-dent on the presence of cells expressing HLA class II

molecules.

*Correspondingauthor.

MATERLILSANDMETHODS

Study participants. Patients 1 and 2 hadpreviously partic-ipated in a study to determine the frequency of

HIV-1-infectedcirculating CD4+ T cells and were selected asthe

two participants with the highest proportions of infected

cells (3). Patients 3 and 4 were randomly selected among

patients attending an outpatient clinic for HIV-1-infected

individuals. They were all free of clinical signs and

symp-toms atthe time of the study (Centers for Disease Control groupII; CD4+ T cellcounts, 0.38 x

109

to0.64 x

109

per

liter). Blood from HIV-seronegative donors was obtained

from the Red Cross BloodBank, Oslo,

Norway.

MAb and immunomagnetic beads. Monoclonal antibodies

(MAb) specific for CD4 (clone 66.1) andtheT-cellreceptor

ac13 heterodimer (clone T10/B9)were generous gifts from J.

Hansen, Fred Hutchinson Cancer Center, Seattle, Wash.,

and J. S. Thompson, University of Kentucky Medical

Cen-ter, Lexington, Ky., respectively. Anti-HLA class IIMAb

HKB-1 and anti-HLA DR MAb B8.11 were generously

providedby S.Funderud, The Norwegian RadiumHospital,

Oslo, Norway, and B. Malissen, Centre d'Immunologie,

Institut National de la Sante etde laRecherche

Medicale-Centre National de la Recherche Scientifique, Marseille,

France, respectively. Anti-CD8 MAb ITI-5C2 and anti-CD14

MAb lD5wereproduced inourlaboratory. Anti-CD4-CD8

MAb(Leu 3/2 Simultest) and their irrelevant isotypic control

MAb were purchased from Becton Dickinson, San Jose,

Calif. Anti-CD3 MAb OKT3wasobtained fromOrtho

Phar-maceuticals, Raritan, N.J. Fluorochrome-conjugated goat

anti-mouseimmunoglobulin G and M-y-, ,u-, andlight-chain

antibodies were purchased from TAGO Inc., Burlingame,

Calif. MAbwerecoatedontoimmunomagneticbeads

(Dyn-abeads M-450; Dynal, Oslo, Norway) as described

previ-ously (3,4).

Flow cytometry. Cells were stained for CD4 and CD8 in

two-color fluorescence studies using fluorescein

isothiocy-anate-conjugated Leu-3a and phycoerythrin-conjugated

Leu-2a, respectively. Fluoresceinisothiocyanate-conjugated

5924

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TABLE 1. Proliferationand HIV-1 production by PBMC depleted of CD8+ T cells in response to activation by SE or T-cell mitogens

Resultfor cells from

patientA:

SEorT-cell 1(1:8,000;1.4x 106) 2 (>1:500; 1.6 x 106) 3(1:20,000;0.9 x106) 4 (1:70,000; 0.84x 106)

mitogena

Prolifera- Day of Peak Prolifera- Day of Peak Prolifera- Day of Peak Prolifera- Day of Peak

tion(cpm)c onset' concn' tion (cpm) onset concn tion (cpm) onset concn tion (cpm) onset concn

SEA 14,200 19 188 5,376f 3 1,045 22,667 - - 10,030 -

-SEB 8,428 19 169 5,357 3 1,225 13,711 - - 6,721 -

-SEC1 10,043 15 439 7,548 3 1,548 11,175 26 1.1 7,588 -

-SEC2 5,878 19 393 7,910 3 1,441 14,061 - - 6,272 -

-SEC3 6,540 23 1.1 4,897 3 1,628 14,412 26 7.6 7,633 -

-SED 40,510 15 329 47,864 3 1,228 27,440 19 1,161 40,185 19 742

SEE 30,922 8 353 41,727 3 1,608 34,914 26 3.5 37,875 23 191

TSST-1 17,845 15 163 6,993 3 734 24,081 - - 7,818 -

-ET 11,200 15 1,383 6,267 3 814 12,310 19 1,519 7,972 -

-MAb OKT3 ND ND ND 16,363 3 677 32,387 12 461 12,944 -

-PHA 7,789 - - 5,214 7 18 15,784 30 4.3 4,283 -

-None 2,213 19 0.1 460 7 0.5 2,120 - - 2,978 -

-aTSST-1,toxicshock syndrometoxin1; ET,exfoliative toxin;PHA,phytohemagglutinin; none,RPMI medium with fetalcalfserum and notoxin.

bNumbers inparenthesesareratios of infectedtouninfectedCD4+ T cells and numbers of cellsperwell.-,negative; ND,notdone. c Measuredas

[3Hlthymidine

incorporation.

dFirst day ofsupernatantHIVp24positivity.

PeaksupernatantHIVp24concentration,innanogramspermilliliter.

fCells were cultured in theabsence ofrIL-2.

anti-HLA DR MAb B8.11 wasused in single-color

fluores-cencestudies. Cellswereanalyzed byflowcytometry

(FAC-Scan; Becton Dickinson, Mountain View, Calif.). The

sig-nals were collected and stored as list mode filesand were

analyzed with the LysisII program.

Cell cultures. Cell subsets were depleted fromperipheral

bloodmononuclear cells(PBMC)by usingimmunomagnetic

beads coated with therelevant MAb, at aratio of 10 beads

percellasdescribedpreviously (9). Following depletion,the

remaining cells always contained less than 2% of the

de-pleted subset. Cells were established as single cultures in

24-well plates (Costar, Cambridge, Mass.) or as triplicate

cultures in 96-well plates in RPMI 1640 (GIBCO, Paisley,

Scotland) containing 10%

(vol/vol)

fetal calfserum

supple-mented with 10 U of recombinant interleukin-2 (rIL-2)

(Amersham International, Amersham, England)perml. The

cellswereactivatedbyanSE(Toxin Technology, Sarasota,

Fla.) at1 ,ug/ml, bythe anti-CD3 MAb OKT3at1

,ig/ml,

or

byphytohemagglutinin at5 ,g/ml.The culturemedium was

changedtwice aweek,and thesupernatantsweretestedfor

the concentration of HIV-1 p24 antigen by means of an

antigen capture enzyme-linked immunosorbent assay (Ab-bott). Thecellswererestimulated bythe original activating

agent atthesameconcentration once aweek. Thecultures,

except those from patient 2, were maintained for 30 days.

SupernatantHIV-1p24inthe cultures of cells frompatient2

peakedondays7through 11,and therefore the cultureswere

terminated onday14. Cell proliferationwas determinedby

transferringvolumescorrespondingto105 cellsaccordingto

the initial cell count to 96-well plates on

day

3,

incubating

with

[3H]thymidine

(2 ,uCi) for 24 h, and then determining

the amount of incorporated radioactivity. In some

experi-ments, separate triplicate cultures containing5 x 104 cells

each were established for the isolation of HIV and for

[3H]thymidine

incorporation.

Limiting dilution assays. For the determination of the

frequencyofHIV-1-infectedcells, CD4+ Tcellswere posi-tivelyselectedby using Dynabeadscoated with theanti-CD4

MAb66.1at aratio of four beads per cell

(3).

The cellswere

separated from the beads byincubationwith Fab

anti-serum (DETACHaBEAD; Dynal), washed twice, and left

overnightat37°C.Seriallydilutedpatient CD4+T cellswere

cocultured with CD4+ T cells from HIV-seronegative

do-nors,andthe cellswereactivatedwith beads coated with the

anti-T-cell receptor MAbT10/B9at aratio offour beads per

cell in the presence of 10 U of rIL-2 per ml as described

previously (3). On the basis of the Poisson distribution

formula, the frequency of infected cells is given as the

number of cells per well required to give 63%

HIV-1-producing wells(18).

RESULTSANDDISCUSSION

PBMCweredepleted of CD8+ T cells in ordertoremove

the cellsresponsible for the suppression of HIV-1 replication

whileretainingHLAclassII-expressingcells (4, 22).These

cellswereactivated in culturebySEA,SEB,

SECi

through

-3, SED, SEE, toxic shock syndrome toxin 1, exfoliative

toxin,MAbOKT3,orphytohemagglutinin. With the

excep-tionof exfoliativetoxin, afinalconcentration of 1 ,ug of toxin

permlwaswell above thesaturatingconcentrationrequired

for theproliferationofPBMCdepletedofCD8+Tcells(data

not shown). Asummary of the results isgiven in Table 1. Themostimportant determinant for the detection ofHIV-1

p24 in the culturesupernatantswasthefrequency ofHIV-1

provirus-containingCD4+ T cells. Thus, HIV-1 replication

was induced by all the SE in cultures of cells from thetwo

individuals with the highest frequencies of HIV-1-infected

CD4+ T cells (patients 1 and 2). When the proportion of

CD4+ T cells infected

by

HIV-1 was

low,

only

those

exotoxins which gavehigh proliferativeresponseswereable

toinduce HIV-1expression (patient4).The timerequiredfor

culture supernatants tobecome HIV-1 p24 positivevaried

considerablyand seemedtodependmainlyonthefrequency

of HIV-1 provirus-containing CD4+ T cells and on the

proliferative response toeach of the staphylococcal

super-antigens. However, the timerequiredfor culture

positivity

could also depend on the phenotype of the HIV-1

quasi-species infecting each individual

(17).

Patient

2,

whose

exotoxin-stimulated cell culture supernatantswereall HIV-1

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A

CD4+LY 110O

CD4- LY

FORWARDSCATTER

B

FORWARD SCATTER

e

cm

0

l

~c-v

I

-w

w we

FORWARDSCATTER

D

FORWARD SCATTER

Iooo

WU-10

RPMV 0.1 FCS

1.0 10 100 1000

§

0 0

40 X a 1. 40

10

M-Concentration of SEDorSEE(nanogram/ml)

FIG. 2. Cell proliferation and HIV-1 production in response to different concentrations of SED and SEE. PBMCdepleted of CD8+ T cells from patient 2werecultured in the absenceofrIL-2.0 and

[l, proliferationinresponsetoSED andSEE, respectively. * and *,supernatantHIV-1p24 concentrationsonday 7 inresponseto SED andSEE, respectively. Inunstimulatedcells, the p24 concen-trationwas0.2ng/mlandtheproliferativeresponse was235cpm(*). FCS, fetal calfserum.

b100

E

n

CD

0u 1 0

N

01

coI

0 E

.P

c

0 0 0

E

I

A B C D

FIG. 1. Results of flow cytometric studies (a) and HIV-1 p24 concentrations in cell supernatants on day 8 (open bars) and [3H]thymidine incorporation (hatched bars) (allvaluesaremedians

andrangesoftriplicateexperiments) (b).PBMC frompatient2were

depleted of CD8+T cells(A),CD8+andCD4+T cells(B),CD8+T cells and monocytes (C), and CD8+ T cells and HLA class

II-expressingcells(D).The cellswereexaminedbyusinganti-CD4 and anti-CD8 MAb in two-color fluorescence studies, butthe popula-tionsarepresented hereas functionsofanti-CD4MAbstaining (y

axis) and size (forward scatter) (x axis).

p24 positiveonday 3, has been extensivelystudied. Allbulk

virus isolates as well as each of several viral clones have

replicatedincell lines and inducedcytopathologyconsistent

with arapid-high phenotype (la).

In order to identify the cell subsets involved in the

SE-mediated induction of HIV-1 expression from PBMC

depletedofCD8+Tcells, cell subsetdepletion experiments

with cells from patient 2 were performed. The results are

presentedin Fig. 1. WhenCD4+ T cellsweredepleted, cell

proliferation wasconsiderably reduced, and HIV-1

expres-sion was completely abrogated (compare the results from

experimentsB andA). Incontrast, depletion ofmonocytes

left the expression of HIV-1 unchanged and caused only a

slight reduction of [3H]thymidine incorporation (compare

theresults fromexperiments C andA). The results show that

thesourceofexpressedvirus intheseexperiments wasthe

CD4+Tcells.Theoretically, monocytescould have

contrib-uted to the concentration ofHIV p24 in the cell

superna-tants. Monocytes have been shown to contain HIV-1 in

some individuals, and the binding of SEto their HLAclass

II molecules has been shown to cause transcriptional

acti-vation of certain genes (21). Experiment C demonstrated,

however, thatmonocyteswereexpendablebothas asource

of HIV-1 andas asourceof HLA classII.Theimportance of

thepresenceof cellsexpressingHLA class II moleculeswas

demonstrated in experiment D. In this experiment, HLA

class II-expressing cells were depleted, removing all the

monocytesandleavingless than0.2% HLADR-expressing

lymphocytes. Both cell proliferation and the expression of

HIV-1 were completely abrogated, although CD4+ T cells

werepresent at aproportion comparable tothose in

exper-imentsCand A (19versus 17% inexperimentC and 9% in

experimentA).Thus, free solubleSEwereunabletoinduce

[3H]thymidine incorporationand theexpressionof HIV-1 in

theCD4+Tcells.

Long-term T-cell cultures require exogenous IL-2. We

tookadvantageof therapidkinetics of HIV-1productionin

cells from patient 2 to examine whether SE might induce HIV-1expression inthe absence ofexogenouslyaddedcell

growth factors. No differences in HIV-1 p24concentration

between cell cultures established with and without

exoge-nous IL-2 were found (the results of the experiment

per-formed in the absence of rIL-2are presentedin Table 1).

Subsequent experiments involving cells from several more

HIV-infected individuals have shown that theproliferative

responseof PBMCdepletedofCD8+Tcells isnotchanged

by the addition of rIL-2 and that SE-activated PBMC

de-pleted of CD8+ T cells from uninfected donors produce

supersaturatinglevels of IL-2 (5).Thissuggeststhat

endog-enousIL-2produced bytheCD4+T cells inresponsetoSE

is sufficienttosustain these cellsthrough2weeks of

contin-uousHIV-1production invitro.

Dilutions of SED and SEE as low as 100 pg/ml still resulted in cell proliferation and highlevels of HIV-1

repli-cation(Fig. 2). In celltitrationexperiments, thefrequencies

of HIV-1 provirus-containing PBMC depleted of CD8+ T

cells were found to be 1:2,000 for SED-responsive and

1:3,000 for SEE-responsive cells (Table 2). Assuming that

these exotoxins activate 20% of the CD4+ T cells at most

a

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TABLE 2. Limiting dilution analysis of the occurrence of HIV-1-producing cells in cells from patient 2

No. ofpositive cultures/totalno.a

No. of

cells/well SED SEE CD4+b

(1:2,000) (1:3,000) (>1:500)

8x 102 0/4 0/4 10/10

4 x 103 4/4 3/4 10/10

2 x 104 4/4 4/4 8/8

1 x 105 4/4 4/4 NDC

aPBMC depleted of CD8+ T cells wereactivatedby SED or SEE in the

absenceof rIL-2 and allogeneic feeder cells. Numbers inparentheses are

resultsespressed as ratios of infected to uninfected cells.

bFordetails of limiting dilution analysis of HIV-infectedCD4+ Tcells,see

MaterialsandMethods.

cND,notdone.

(12),thisindicates that each exotoxin-binding, HIV-1

provi-rus-containing cell was induced to support active HIV-1

replication. This conclusion is supported by the results of the

experiments using cells from patient 4. PBMC depleted of

CD8+Tcellsusuallycontain less than50% CD4+ T cells (2).

Ifthis was the case also for patient 4, then SED and SEE

induced HIV-1 expression in wells containing fewer than 6

HIV-1provirus-containing CD4+ T cells (Table 1).

Our results contrastwith those in arecent article, which

reportedthat the PBMC depleted of CD8+ T cells from all

asymptomatic HIV-1-infected individuals studied failed to

proliferate and instead showed evidence of death by

apop-tosis in response to activation by SEB (10). That study

involvedalarge number of HIV-infected individuals, and the

methods employed werequitesimilar to the ones used in the

present study. Although this study involved only a small

groupofHIV-1-infected individuals,weshow by

[3H]thymi-dineincorporationthatPBMCdepleted of CD8+ T cells are

capableofproliferation, andbythemeasurement of

super-natantp24 concentrationweshowthat these cellsareableto

continuouslyexpress HIV-1 forat least 3 weeks in culture.

Cell numbers were greatly increased in these long-term

cultures (datanotshown). Insubsequent studies, involving

other HIV-1-infected individuals, we have found that the

SE-induced

proliferation

of PBMCdepletedofCD8+Tcells, measured as [3H]thymidine incorporation (counts per

minute)per CD4+ Tcell present in thewell,was as strong

for cells from the HIV-1-infected studyparticipants as for

cells from HLA DR- and age-matched uninfectedcontrols

(2).Thediscrepancyinresults between thetwostudiesmust

beleftunexplainedatthe presenttime.

One unexpected observation made during these

experi-ments was that SED and SEE gave considerably higher proliferativeresponsesthan theother exotoxinsormitogens (Table 1), suggesting binding by SED and SEE to a

high

proportion of T cells in our patients. This contrasted with

resultsobtainedbytestingPBMCdepletedofCD8+ Tcells

obtained from nineHIV-1

seronegative donors,

where SED

and SEE gave proliferative responses similar tothe other

exotoxinsandmitogens

(data

not

shown).

Inother studies of

HIV-1 seronegative individuals, neither SED nor SEE has

been showntobindtoa

disproportionately large

fraction of

T-cellreceptor

VP-expressing

cells

(6,

16).

The

significance

of theseobservations remains tobeestablished.

Certain microbial gene

products,

notably

from

cytomega-lovirus,Epstein-Barr virus,and humanT-cell leukemia virus

type I

(HTLV-I),

have been showntotransactivate HIV-1

longterminal repeat(7, 13,

19). Theoretically,

such

transac-tivating agents might bypass the requirement for cell activa-tion by specific antigen in order for HIV-1 producactiva-tion by infected cells to occur. However, these agents either are rare (HTLV-I) or require coinfection of the same cell for trans-activation of HIV-1 to occur (Epstein-Barr virus and cyto-megalovirus). They are thus likely to be of limited

impor-tance ascofactors for thedevelopment of AIDS. In contrast,

staphylococci are common pathogens, and more than 40% of all strains produce one or more exotoxins (8, 14). Our experiments demonstrate that these exotoxins, at very low concentrations and without exogenously added cell growth factors, can induce virus expression from each

HIV-1-infected,superantigen-responsive CD4+ T cell in vitro. Such

superantigenic T-cell activation may also induce HIV-1 replication in vivo and thus may represent an important cofactor for the development of AIDS.

ACKNOWLEDGMENTS

Weare indebted to M. Sl0rdahl, Section for AIDS Prevention,

Oslo City Department for Health and Environment, and her pa-tients. WethankJ. Hansen, J. S. Thompson, B.Malissen, and S. Funderud for kindgifts of MAbs. We also thank T. Egeland for helping with the flow cytometry studies.

This study wassupported by the Norwegian Cancer Society.

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