Varicella-zoster virus-specific cytotoxic T lymphocytes (Tc): detection and frequency analysis of HLA class I-restricted Tc in human peripheral blood.

0022-538X/87/113463-07$02.00/0

Copyright C) 1987, American Society for Microbiology

Varicella-Zoster

Virus-Specific

Cytotoxic T Lymphocytes (Tc):

Detection and Frequency Analysis of

HLA

Class

I-Restricted

Tc

in Human

Peripheral

Blood

JULIAN K. HICKLING, LESZEK K. BORYSIEWICZ, AND J. G. PATRICK SISSONS*

MRC Clinical Immunology ResearchGroup, Royal Postgraduate MedicalSchool, London W12 OHS, United Kingdom Received 24June1987/Accepted4August 1987

The cytotoxic T-cell (Tc) response to varicella-zoster virus (VZV) is incompletely characterized. We investigated whether VZV-specificTc restricted by class I products of the majorhistocompatibilitycomplexcan

begeneratedfrom theperipheral blood of VZV-immune donors. Cell lineswere established from peripheral

bloodlymphocytes (PBL)of seropositive donors by secondary in vitro restimulation. If cell-freeVZVwasused

asthestimulating antigen, theresulting lineswerepredominantly CD4+ and didnotshow class I-restricted cytotoxicity; whenautologous infected fibroblasts wereusedfor in vitro stimulation, the resultant lineswere

usually cytotoxic,although inonly4of11subjectstestedwasthiscytotoxicityHLArestricted and virus specific.

PBL were also tested for Tc activity without prior restimulation; VZV-specific Tc activity was only

demonstrable in thePBLofasubjectconvalescentfollowingzosterbutnotfrom subjects withrecentvaricella infectionorfrom normal subjects. VZV-specificTcprecursorfrequencieswerethendetermined in six selected

subjects bylimiting-dilution analysis. Ameasurable frequency was detectable in four of thesix seropositive

subjects, ranging from 11/106T cells inan asymptomatic carrier, to63/106 T cells ina subject with recent zoster.Weconcludethatvirus-specific majorhistocompatibility complex class I-restrictedTcprecursors may

be present intheperipheral blood of normal individuals seropositive forVZV butatafrequencylower than

thatforotherherpesviruses with nonneuronal sites of latency.

Varicella-zoster virus (VZV) is an alpha herpesvirus of humans. Primary infection may be associated with the

clinicalsyndrome ofvaricella(chickenpox), followingwhich thevirus establisheslatentinfection insensory nerveganglia

(7). Reactivation may subsequently occur and is associated

with dermatomallesions (zoster). An increased frequency of virus reactivation and greater likelihood of disseminated

infection are both associated with acquired or inherited

defects ofcellular, rather than humoral, immunity (12). It may thusbe inferredthat cellularimmune mechanisms are

important in controlling both primary infection by and

reactivation ofVZV.

Aknowledge ofthe natureoftheT-cell response to VZV

would aid understanding ofhow viruses establishing classi-callatency withinthenervous system maybecontrolledby the immune response. Theproliferative response of mono-nuclearcellstoVZVantigenshaspreviouslybeen studiedas a measure of T-cell immunity and has been shown to

increase during recovery from varicella (1, 10) and zoster

(15, 19). However, in comparison with the other human

herpesviruses,thecytotoxicTcell(Tc)responsetoVZV has

receivedrelativelylessattention.Tccanbedetected follow-ing infection with other human herpesviruses and are be-lievedtohavearole inlimiting reactivationepisodes during persistent infection. By using

techniques

of secondary in vitrorestimulation, class I-restricted Tclinescan be gener-atedfromperipheral blood

lymphocytes

(PBL)of

asympto-matic subjects

seropositive

for human

cytomegalovirus

(HCMV),Epstein-Barrvirus(EBV),orherpes

simplex

virus

(HSV) (2, 18, 21); HLAclass I-restrictedTc are

reportedly

directly detectable during HCMVinfection, and their

pres-* Correspondingauthor.

ence may be associated with survival from infection by marrowtransplant recipients (16).

More recently, Hayward et al. (9) have reported the

generation in vitro ofCD4+, class II-restricted Tc clones

specificfor VZV. SimilarCD4+ Tc clones specific for HSV havepreviously beengeneratedand have been showntobe

specific forHSVglycoproteins, in particular glycoproteinD (22).

Previous worksuggests that in general CD8+ HLA class

I-restricted Tc precursors (Tc-P) appear to be the

numeri-callypredominantTcpopulationin humanperipheral blood,

asshownby limiting-dilution analysis againsttargetcells in the presenceof lectin(13). Wethereforeset out todetermine

whetherCD8+, HLA class I-restricted VZV-specific Tc, or Tc-P could be demonstrated in the peripheral blood of

asymptomatic,

VZV-seropositive subjects. We report

meth-ods by which VZV-specific Tc may be generated in bulk culture fromsome but notall seropositive donors. In

addi-tion, by usinglimiting-dilution analysis,weobtained

prelim-inary estimates ofthe frequency ofVZV-specific Tc-P in

normal subjects and in those recovering from zoster and found that the frequency of

VZV-specific

Tc-P was low

relativeto thatfor herpesviruses with nonneuronal sites of latency.

MATERIALSAND METHODS

Virusstocks. Plaque-purifiedEllen strain VZV(American

TypeCultureCollection, Rockville, Md.)wasgrownin Flow 5000 humanembryo fibroblasts

(Flow

Laboratories,

Irving,

Scotland) by

passaging

infected

monolayers

showing

75%

cytopathic effect onto fresh

monolayers

at a ratioof 1:5 of infected to uninfected cells. VZV stocks were stored in

liquidnitrogenascell-associatedvirusandwereshowntobe

mycoplasma free by culture and Hoechst

dye (no.

33258)

3463

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staining. Plaque-purified HSV and CMV (ATCC AD169) stockswereharvestedfrom thesupernatant of infectedFlow 5000monolayersandstoredat -70°Cascell-free virusat106 to

107

PFU/ml.

Human fibroblast lines. Primary human fibroblast lines

wereestablishedfrom skinbiopsiesfromHLA-typeddonors as described by Borysiewicz et al. (2). Primary fibroblasts

were grown in Eagle minimum essential medium (Flow)

supplemented with10% fetal calfserum(Imperial

Laborato-ries), 1% nonessential amino acids (GIBCO Ltd., Paisley, Scotland), 2 mM L-glutamine, 105 IU ofpenicillin perliter, and 100mgofstreptomycin (Flow)perliter.

PreparationofT-cell lines. Donors werehealthy

seroposi-tive individuals asjudged by a titer of complement-fixing

antibody to VZV ofgreater than 1 in 8 and/or lymphocyte

proliferationinresponsetoVZV.Peripheralblood

mononu-clearcells(PBM)wereobtainedby Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) gradient centrifugation andsuspendedat106/ml in 24-wellplates (BectonDickinson Labware, Oxnard, Calif.) in RPMI 1640 (Flow) with 10% heat-inactivated human AB serum. The stimulating antigen

was either 5 x 104autologous VZV-infected fibroblastsper

well (autologous or partially matched for HLA A and B antigens) or cell-free VZV antigen. Cell-free VZV antigen was prepared by sonication of infected fibroblasts(showing greaterthan 90% cytopathic effect)followedby heat inacti-vation at 56°C. This wasused ata concentration shown to induce proliferative responses as measured by tritiated thymidine uptake in preliminary experiments. Interleukin 2 (IL-2;CellularProducts, Buffalo, N.Y.)wasaddedonday3 (or day7 inearly experiments) andevery subsequent3 or4

days. Fresh antigen was added on day 7, with autologous irradiated(3,500rads) PBMasfeeder cells(106/ml). After 7 to 26 days invitro, cells were harvested, washed, and used

aseffector cells ina6-h51Cr-releaseassay.Thephenotypeof the cells wasdetermined byflowcytometry.

Cytotoxicity assay. Target cellswere autologous (orHLA A- and B antigen-matched) and HLA-mismatched fibro-blasts,used eitheruninfectedorinfectedwithcell-associated VZV(1:5ratio ofinfectedtouninfectedcells)72 hbefore the

assay.Targetcellswerelabeledwith51Cr(200 p.Ci/106cells;

Amersham International, Amersham, England) overnight, trypsinized, washed thoroughly, and suspended to 5 x

104/ml. Target cells(100 pulperwell) wereadded to96-well round-bottom microtiterplates (Becton Dickinson) already containing 100 pl of effector cells, giving the required effector-to-target-cellratiosintriplicate. After 6h, 100 of supernatantwasharvested,andradioactivitywascounted in

a gamma-counter (LKB Wallac Multigamma, Turku, Fin-land); specific chromium release was calculated by the standardformula. Maximumrelease wasobtained by using 1% TritonX-100. Average spontaneous release from VZV-infected target cells was 22.5% of maximum in 6-h assays and 30% of maximum in 18-h assays. At least 70% ofthe VZV-infected target cells were always positive by indirect

immunofluorescence by using zoster-immune globulin (Cen-tral Public Health Laboratory Service, Colindale, England) and fluorescein-conjugated rabbit anti-human immunoglob-ulinG, A, andM, kappaand lambda(Dakopatts, Glostrup, Denmark).

Limiting-dilution analysis. PBL obtained from Ficoll-Paque centrifugation were incubated with AET (Sigma Biochemicals, Poole, England)-coated sheep erythrocytes (Tissue Culture Services, Slough, England) (4), and the AET-E+ population was collected after centrifugation on

Ficoll-Paque and suspended in RPMI 1640 with 10% fetal

calfserum atconcentrationsrangingfrom 5 x 105/mlto 1 x

104/ml. Cellsuspension (100

RI)

wasadded toround-bottom microtiter

wells,

with 30 replicate wells at each concentra-tion. Each well alsocontained 1 x

105

irradiated

autologous

PBM asfeeder cells and 2 x

103

VZV-infected

autologous

fibroblasts. Inone

experiment, lymphocytes

wereincubated

with concanavalin A (5 ,ug/ml) for 48 h and then used as

responder cells in the absence of antigen. This

polyclonal

stimulationprotocolresulted infrequencies comparablewith those from antigen stimulation in control experiments. Su-pernatantfromMLA 144 cells wasusedas a sourceof IL-2 and was present from day 0. Cultures were refed every 3

days and maintained for 9 to 12 days. At the end of this

period, each well was split into four aliquots for testing againstfour targets ina12-h51Cr-releaseassay.Targetsused were K562in the presence ofphytohemagglutinin (PHA)(4 pug/ml), VZV-infectedautologous fibroblasts, CMV-infected

autologous fibroblasts, and uninfected autologous fibro-blasts. A positive result against a given target was definedas aresult greater than 3 standard deviations above spontane-ous release obtained in the presence of target and feeder cells only. The number of positive wells mediating lysis of VZV-infected targets and K562 plus PHA but not control targetswasdetermined for each concentration. The propor-tion ofnegativewellsateach concentrationwasdetermined and plotted on alog scale against input cell number per well. The frequency was calculated from the input cell number giving 37% negative wells. Tc-P frequencies were calculated by using least mean squares regression analysis(correlation

coefficient, r > -0.9 in most cases) (11) and maximum likelihood analysis (5). There was no significant difference between the results obtained by each method.

Determinationof cellphenotype. Aliquots of effector cells were incubated for 45 min with anti-Tll, -T8, -T4 (Coulter

Electronics Ltd., Luton, England) or Leu-7 (Becton Dickinson) at a 1/40 dilution, followed by the addition of

fluorescein-conjugated goat anti-mouse immunoglobulin (Coulter). Analysis was performed on an EPICS-C flow cytometer.

RESULTS

Secondary in vitro stimulation with cell-free VZV. Data from other workers (1, 10, 15, 19) and preliminary

experi-ments performed by us show that PBL from subjects

seropositive for VZV proliferate in response to cell-free VZV antigen preparations (data not shown). Experiments wereperformedtodetermine whether therespondingcells in these cultures were capable oflysing VZV-infected fibro-blasts.

PBL from healthy seropositive subjects were cocultured with heat-inactivated cell-free VZV and expanded in the presence of IL-2. Cultures weremaintainedfor 7 to 26 days before being tested in a 6-h 51Cr-release assay. Lines from seven donors were tested, but no cytotoxic activity was observedagainst autologous ormismatched VZV- or HSV-infectedfibroblasts (data not shown). The phenotype of the

respondingcells wasgreater than80%CD2+, and the ratio of

CD4+ toCD8+ was alwaysgreater than 2:1 (Table 1).

Secondary in vitro restimulation with infected autologous fibroblasts. Twomajor possibilities, notmutually exclusive, could explain the failure to generate VZV-specific HLA class I-restricted Tc by cococulture with cell-free antigen.

First,itmightbe thatantigen isnotpresented in the context ofappropriate major histocompatibilitycomplex antigens, or

second, theappropriate VZV antigens required to generate

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TABLE 1. Phenotypes ofshort-term T-cell bulk cultures established in response to VZV

Stimulating antigen %Positive (range)

CD4+/CD8+b

(no.of subjects) CD2+ CD4+ CD8+ Leu-7

Cell-free VZV (n = 6) 88.8 (84-96) 60.7(50-81) 18.5 (6-25) 5.6 (2-11) 4.98 + 4.7

VZV-infected fibroblasts (n = 10) 84.8(72-97) 28.9 (15-46) 46.6 (30-60) 5.6(2-8) 0.63 ±0.23 aExpressed as mean percent of positive cells.

bMeanratio of CD4+ to CD8+ + 1 standard deviation.

Tc invitro mightbe absent fromthecell-free virion prepa-ration. In view of ourpreviousexperiencewithCMV (2), the latter possibility was investigated by using VZV-infected

autologous fibroblasts as the stimulating antigen; these wouldcontain nonstructuralVZVproteins, absent from the

cell-free preparation. IL-2 wasaddedonday 7 of culture(in

later experiments, the addition of IL-2 on day 3 made no

difference intheresults).

In contrast to theresultsobtained from lines generated in

response to cell-free antigen, cultures stimulated with in-fected fibroblastswerepredominantly CD8+ (meanratioof CD4+ to CD8+, 0.63) (Table 1). The cytotoxic activity observed in 11

asymptomatic

seropositive donors could be

dividedinto threegroups(Fig. 1).Lines from4of11donors

showed HLA class I-restricted cytoxicity against

VZV-infected targets (JH, PS, DS, and MF). Linesfrom donors

TA, JE, JM, and SG displayedcytotoxic activity, although

this was not HLA restricted. Finally, lines from a further three donors (HB, BR, and LB) displayed little or no

cytotoxicity. Inrepeatexperiments, T-cell lines established fromanindividualsubjectgenerallygave the same patternof

response.VZV-specific, class I-restrictedTc lines have been

consistently generated from donor JH (7 of10lines

gener-atedshowed class I-restricted cytotoxicity).

To confirm the virus specificity ofthe responding cells, lines from two donors (JH and PS) which showed

HLA-restricted cytotoxicity against VZV-infected targets were

also tested against autologous fibroblasts infected with HSV (Fig. 1). HSV-infected autologous fibroblastswerenotlysed above background levels.

Studies on subjects with recent varicella or zoster. The

failure to generate VZV-specific Tc lines from some

seropositive donors mightbe due to alow frequency of Tc-P cells in these individuals. To determine whether the fre-quencyof VZV-specificTc wastemporallyrelated torecent

primary infection orreactivation, donors who hadrecently experienced

varicella

or herpes zoster were tested. Donor MF had varicella 12 months before testing, and donorsTA and HB had varicella 2 months before testing. When their PBL were stimulated with infected fibroblasts, the results

did not differ significantly from those obtained in other seropositive subjects (Fig. 1). A Tc response was obtained from MF,although notfrom the subjectswith more recent

infection (TA and HB).

Inaddition,PBLfromthesesubjectsweretesteddirectly in

51Cr-release

assays without prior secondary in vitro restimulation (Fig. 2). There was no significant lysis at 6h,

and therefore the results from the 18-h assays are shown. Four donors with recent varicella (TA, MH, HB, and EA)

showednopreferential lysis of VZV-infectedtargetssharing

one or two HLA class I A and B antigens (autologous fibroblasts were not available) compared with

HLA-mismatched fibroblasts. Similar results were obtained with

asymptomatic seropositive subjects with no recentepisodes of varicella or zoster. The

nonspecific lysis

observed with donorEA(whohadvaricella2monthspriortoassay)could be reduced by rosetting the effector population with ox

erythrocytes coated with rabbit ox erythrocyte anti-body. The remainingcellpopulation mediated virus-specific HLA-restricted lysis(Fig. 3).

PBL from two subjects with recent episodes of zoster werealsotestedwithoutpriorrestimulationinvitro, i.e., PC

and SB, 3 months and 4 weeks, respectively, after their initialpresentation. Bothhadincreasedlevelsof CD8+ cells

40.

30-20)

0J

Donor JH PS DS MF TA JE

.JM

SG HB BR LB

FIG. 1. Cytotoxicityexpressed byT-cell linesestablishedinresponsetoinfected fibroblasts. Culturesweretestedonday14. Resultsare expressedasspecific 51Cr release obtainedat aneffector-to-T-cell ratio of 20:1(+ 1 standarddeviation)in a6-hassayfrom thefollowing targets. F, Autologous (or partially HLA class I-matched) VZV-infected fibroblasts; i, Autologous uninfected fibroblasts; g,

HLA-mismatched,VZV-infectedfibroblasts; [1, autologousHSV-infectedfibroblasts.

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30 w

u 2

O10

OlS

AT-

SIV

Donor CC TA MH EA HB PC SB

FIG. 2. Cytotoxicity

produced directly by

fresh PBL from donors withrecentvaricellaorzoster. Barsrepresent

51Cr

release inan 18-h

assayat aneffector-to-T-cell ratio of 20:1 (+1 standard deviation). Subjects TA, MH, EA, andHB weretested2months afterpresentation with varicella. Subject PC was tested 3 months after presentation with zoster. Subject SB was tested 3 weeks afterpresentation with zoster. Subject CG is anasymptomaticseropositive control. For explanation of bars,seelegendtoFig. 1.

intheirPBL(CD4+ toCD8+ ratio of 0.5:1 in both cases as determined byflowcytometry). Donor SB showed no

pref-erentiallysis of VZV-infectedtargetssharingtwo HLAclass Iantigens compared with completely HLA-mismatched con-trols. In contrast, PBL from PC exhibited class I-restricted

lysis of VZV-infected fibroblasts. HSV-infected autologous

fibroblasts matched at three class I loci and

HLA-mis-matched fibroblasts were not lysed, suggesting that

VZV-specificTc were present in PBL at a sufficientfrequencyto

be detected without invitrorestimulation.

Limiting dilution analysis ofVZV-specific Tc-P frequency. To measurethefrequency ofVZV-specific Tc-Pin different individuals and compare it with those for other human

herpesviruses, we used the technique of limiting-dilution analysis as described in Materials and Methods (and by

L. K. Borysiewicz, S. Graham, J. Hickling, C. Green, and J. G.P. Sissons [submitted for publication]).

40

-as

In -) 30

-L.

i

20-u

m10_

uI

0-O

-IIt

J .

-1 I

-- I

A

Theability of cells from each well in the assay to mediate

lectin-dependent cytotoxicitywas used to measure the fre-quency of all cytotoxic cells regardless oftheir specificity

and was between 67 and 500 per 106Tcells. Results from a

typical experiment are given in Fig. 4, and a summary of

results from sixsubjects isgivenin Table 2. In all cases, the

frequency of Tc-P mediating specific lysis of autologous

uninfected and CMV-infected fibroblast target cells was low orundetectable. In twoasymptomatic seropositivesubjects

(SH and AR), the VZV-specific-TcP frequency was no greater than that for uninfected target cells. However,

de-tectable frequencies of 11 and 29 per 106 T cells were

obtained from asymptomatic subjects JH and DS,

respec-tively; PBL from both these donors had previously been

observed to give rise to VZV-specific, class I-restricted

cytotoxiclines in the bulk culture system(Fig. 1).

The highest VZV-specific Tc-P frequency (63

VZV-407

30_

50 25 12.5

E:T

6.25

B

I I I

-I

50 25 12.5 6.25

E:T

FIG. 3. Cytotoxicity expressed by fresh PBM from subject EA 2 months after varicella. (A) Unfractionated PBM. (B) After depletion of FcR+ cells. Cells were tested in an 18-h51Cr-releaseassayagainst VZV-infected autologous fibroblasts(0), uninfected autologous fibroblasts

(0),VZV-infected HLA-mismatchedfibroblasts (A), and K562 cells (A). Standard deviations were<10%of meanspecific-release values.

20-

10-oi

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Input

cells/well,

104

0 1.0

.0

t, .6

ci

.

.0 .37-,

C

o

0

L.

IL

.2

2 4 6 8

,e. ,eeseeeS 59

10

1/9x

104

1/4 x

103

FIG. 4. Frequency analysis of VZV-specific Tc-P in

donor

JH. The results fromlimiting-dilution analysis are shown as asemilogplot of results from a representative experiment. After 13 days in vitro, cells from each well were split and tested against K562 plus PHA(a), autologous VZV-infectedfibroblasts (O), and uninfected fibroblasts (A). There was no lysis of CMV-infected fibroblasts. It can be seen that thecalculated precursor frequency cells mediating lectin-dependent cytotoxicity was 1 in 4,000, and for VZV-specific Tc, the frequency was 1in90,000 (250 and 11per 106Tcells, respectively). Lines were plotted by using linear regression analysis.

specific Tc-P per

106

T cells) was detected in PBL from

donor DB, who was tested 3 months after an episode of zosterbutwasotherwisehealthy.Incontrast, donorSBwas

tested 4 weeks afterzoster butwas on immunosuppressive therapyfollowingarenalallograft. Inthisexperiment, PBL

were polyclonally activated with concanavalin A for 48 h

prior to plating out in limiting dilution, as opposed to

stimulation

withVZV-infectedfibroblasts.Thefrequency of

VZV-specific

Tc-Pobtained(29 VZV-specific Tc-P per 106 T

cells)

is

comparable withthehighest frequency observed in anyasymptomatic seropositive subject.

DISCUSSION

Inthisreport, wehavedescribedamethodofsecondaryin vitro restimulation which is capable of generating major

histocompatibility

complex class I-restricted VZV-specific Tc lines from a proportion of asymptomatic seropositive

TABLE 2. Frequencies ofVZV-specificTc-Pderivedfrom limiting-dilution analysis

No.ofspecificTc-Pper106Tcells withtarget:

Donor K562 cells VZV-infected CMV-infected Uninfected

+ PHA fibroblasts fibroblasts fibroblasts

JH 250 11 <2.0 2.5

DBW 100 63 <2.0 <2.0

SH 500 <2.0 <2.0 3.3

AR 115 <2.0 <2.0 <2.0

DS 122 29 <2.0 <2.0

SBb 323 29 <2.0 <2.0

aTested3monthsafterpresentationyvithzoster.

bTested 4weeks after presentation with zoster and receiving

immuno-suppressive therapyattime oftesting.

donors. VZV-specific Tc were only generated if infected

fibroblasts, rather than cell-free VZV, were used as the

stimulating antigen. Thefrequency of such cells, estimated

bylimiting-dilution analysis,wasbetween29 and <2 per 106 cells in thoseindividuals we studied. Furthermore, prelimi-nary results suggest that there is little difference in the response produced by donors 2monthsafteranepisode of

varicella compared with that of asymptomatic seropositive

individuals. However, a recent episode ofzoster may be

associated with an increased frequency of VZV-specific

Tc-Pasmeasured bylimiting-dilution analysis.

The importance ofthevirus preparationused to restimul-atePBLis shownbythegeneration ofpredominantlyCD4+ T-cell lines to cell-free virus and CD8+ T-cell lines to

autologous virus-infected fibroblasts, as previously shown for HCMV(2).Oneexplanation forthis is that nonstructural

virus antigens expressed only in the infected cells are

dominanttargetantigens for Tc,asinthe caseof HCMV(2). Thispossibility is difficulttoinvestigate because the strongly cell-associatednatureof VZVmitigates against synchronous infectionand thesubsequentuseofmetabolic inhibitors such

as phosphonoformate which only permit

expression

ofthe virus immediate-early and early proteins. An alternative

explanation isthat naturalvirusinfectionof stimulatorcells may beimportantifendogenoussynthesisof viralproteinsis

required for their presentation in the context of major histocompatibility class I antigens (6), as shown for the

generationofinfluenza

hemagglutinin-specific

Tc(14). However, evenby

using autologous

infected

fibroblasts,

classI-restricted

VZV-specific

Tclineswere

only

generated

in one-third ofthedonors tested. In the other

subjects,

the

nonspecific cytotoxic

activity

was

presumably

attributable

to natural killer (NK) or

lymphokine-activated

killer cells.

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This variation among seropositive subjects could reflect a

lower frequency of VZV-specific Tc-P in some donors. In

view

of this

possibility,

we

used

limiting-dilution analysis

to

quantitatively

assess thefrequency of VZV-specificTc-P in a small group of seropositive subjects. A measurable fre-quency of

VZV-specific

Tc-P was detected in two of four

healthy

asymptomatic subjects. Both thesesubjects hadalso

given

good responses in thebulk culture system, suggesting

that there maybeacorrelation betweenTc-Pfrequencyand the

ability

to generate Tcin culture. However, these

VZV-specificTc-Pfrequenciesare considerably lower than those

described forotherherpesviruses. Forinstance, ananalysis of HCMV-specific Tc-P frequencies in asymptomatic

sub-jects seropositive

for both HCMVand VZV suggests that the

HCMV-specific

Tc-P

frequencies

are in the range of50 to

200 per 106 T cells, at least fivefold higher than those for

VZV

(Borysiewicz

et al., submitted). In addition, the fre-quency of EBV-specific Tc-P (although estimated by a

different

method,

the

regression

assay) is reportedly in the range of 100 to 1,000 per

106

T cells (17). The highest

VZV-specific

Tc-P frequencies were detected in subjects withrecentepisodes ofzoster(DB and SB). These increased

levels of Tc-P are consistent with recent restimulation in

vivoand suggest that such cells mayplayarole incontrolling reactivation

episodes.

It is possible that the VZV-specific Tc-P

frequency

observed in SB was reduced owing to the

immunosuppressive

therapy the subject was receiving, as

has been

previously

reported for EBV-specific Tc-P

(20).

Owing

to the

difficulty

in

finding patients

with acutezoster

with haplotypes matching those available in our panel of

primary

fibroblast lines, relatively few donors have been

tested to

date,

and none

during

the acute phase of the

disease.

Previousattempts to compare the levels of T-cell

immu-nity

to VZV in

subjects

with varicella or zoster have

focussedon

lymphoproliferative

responses toVZVantigens

(1, 10, 15, 19).

These have shown that the mean in vitro

blastogenic

response declines to low levels within 30 to 50

days

of

eruption

of the varicella rash yet still remains

extremely

high

for thesame

length

of time after the appear-anceofzoster. This would beconsistent

with

ourresultson

cytotoxic

cells obtainedfrom

limiting-dilution

analysis. There are also

previous

reports onthedetection of

VZV-specific

Tc.

Hayward

et al. (9)

recently

reportedthe

gener-ation of

VZV-specific CD4+

Tcclones whicharecapableof

lysing

HLA-DR-matched EBV-transformed B-cell lines

superinfected

withVZV. Itshould be noted thatitispossible

thatthe

CD4+

linesgeneratedin ourexperiments didpossess

cytotoxic potential

but that such activitywas notdetectable

by using primary

fibroblasts astargetcells, as they do not express HLA class II

antigens,

which are the restricting elementfor the

expression

of effector function by CD4+ T cells.

Inanotherstudy, Bowden etal. (3) used a direct

cytotox-icity

assay

system

(without restimulation in vitro) to

inves-tigate lysis

ofVZV-infected fibroblastsby PBL from

asymp-tomatic

healthy subjects.

They found that cytotoxicitywas

enhanced

by

matchingthetarget and effector cells for HLA class I

antigens (in

contrast tothe results we obtained) and was reduced by panningthe effector populations with anti-T8monoclonalantibodies, suggestinga role for CD8+Tc. In

addition,

monoclonal antibody blockingand depletion

stud-ies also indicated involvement of CD4+ and NK cells. Furtherworkby Haywardetal. (8) also suggested that NK cells contribute to the lysis of VZV-infected fibroblasts in sucha systemand that it is difficult to distinguish between

lysis mediateddirectly by Tc andthatmediated byNK cells amplified by lymphokine release from T cells. We

assayed

PBLfrom both normalseropositive donorsand donors with

recent varicella indirect cytotoxicity assays(in addition to assays after secondary restimulation in vitro). PBL from

both groups exhibited non-HLA-restricted

cytotoxicity

when assayed directly. Although we have not performed

detailed studies to determine the phenotype of the cell

population(s) mediating this nonspecific lysis, it was noted that the population responsible couldbe removed by

deple-tion of FcR+ cells, consistent with there being an NK component. Two subjects with recent zoster were also tested in direct cytotoxicity assays without preculture of

PBL, and cells from one of these subjects (PC) mediated VZV-specific, HLA class I-restricted cytotoxicity.

In summary, we demonstrated the presence of major histocompatibility complex class I-restricted Tc-P

specific

for VZV in the peripheral blood ofsome seropositive sub-jects. Our initial results suggest that these Tc-P are present at a relatively low frequency, which may be higher after

reactivation of VZV. It might be supposed that

herpes-viruses exhibiting classical neuronal latency, such as VZV, would produce less restimulation in vivo than those which

persist in lymphoid and epithelial cells, with intermittent

virus production, such as EBV and HCMV, and this could be one explanation for the relatively low frequency of

VZV-specific Tc-P. Further work is needed to define the precise specificity of VZV-specific Tc-P. The use of expres-sion systems containing isolated VZV genes would provide a means of sodefining specificity, and we are currently pur-suing this end.

ACKNOWLEDGMENTS

We are grateful to J. R. Batchelor, Department of Immunology, Royal Postgraduate Medical School, for the HLA typing, and we thank K. Bateson for preparation of themanuscript.

This work is supported by the Medical ResearchCouncil. J.K.H. is in receiptofa Medical Research Council researchstudentship, and L.K.B. isa ListerResearchFellow.

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