INFECTION AND IMMUNITY, Oct. 1977, p. 130-137Copyright © 1977 American Society for Microbiology

Vol. 18, No. 1Printed in U.S.A.

Sequential Changes in Cell-Mediated Immune Responses toHerpes Simplex Virus After Recurrent Herpetic

Infection in HumansE. J. SHILLITOE,t* J. M. A. WILTON, AND T. LEHNER

Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, LondonSEI 9RT, England

Received for publication 17 March 1977

Lymphocyte responses to herpes simplex virus (HSV) were studied in 23patients with recurrent herpes labialis and in 19 control subjects. Lymphocytesof seropositive, but not seronegative, controls responded to HSV by thymidineincorporation, and the supernatant fluids inhibited the migration of guinea pigmacrophages. Lymphocytes from patients with a recurrent herpetic lesion re-sponded to HSV by significantly greater thymidine incorporation than seroposi-tive controls, but supernatants did not show an increased macrophage migrationinhibition response. During the 28 days after the onset of a lesion, the thymidineincorporation to HSV fell to the level of the seropositive controls, and superna-tants then induced an increased inhibition of macrophage migration. Lymphocyteresponses to Candida albicans, purified protein derivative, or phytohemaggluti-nin did not fluctuate according to the presence of a lesion and did not differfrom those of the controls. Lymphocyte responses to HSV were unaffected byculture in the presence of serum from seronegative or seropositive controls, orfrom patients with or without a herpetic lesion. It is suggested that in patientswith recurrent herpes labialis a periodic defect ofthe migration inhibition responsemight have allowed the recurrent infection to develop, and that the increasedthymidine incorporation stimulated by HSV in vitro is a result of antigenicstimulation from the lesion.

Individuals prone to recurrent infection byherpes simplex virus (HSV) have circulatingHSV antibody, and their lymphocytes respondto HSV by increased thymidine incorporation(21, 29). An immune defect was postulated byWilton et al. (29), who reported that superna-tants of HSV-stimulated lymphocytes from pa-tients with a herpetic lesion failed to inhibitmigration of guinea pig macrophages. This wasconfirmed by the finding that leukocyte migra-tion was not inhibited by HSV when the patienthad a herpetic lesion (7). However, in the ab-sence of lesions in susceptible subjects, inhibi-tion of leukocyte migration was found to beintact (23). In the present study sequential ob-servations were made on the thymidine incor-poration and macrophage migration inhibitionresponses to HSV of lymphocytes from patientsprone to recurrent herpes labialis (RHL). Inaddition, the influence of serum factors on thelymphocyte response was studied by culture inthe presence of sera from different phases ofRHL and controls.

t Present address: Department of Microbiology, The Mil-ton S. Hershey Medical Center, The Pennsylvania State Uni-versity College of Medicine, Hershey, PA 17033.

MATERIALS AND METHODS

Patients. A series of 23 patients with RHL wasstudied; the age range was 18 to 46 years (mean 27),and there were 9 males and 14 females. All were ingood health and not receiving any drugs. Immunolog-ical investigations were performed as soon as possibleafter the onset of a recurrent lesion and again 2 and4 weeks later. The diagnosis of RHL was made onthe basis of the patients' history, the appearance ofthe lesion, and virological cultures. As controls, agroup of 19 healthy individuals, matched for age andsex, was studied.Complement-fixing antibody. Serum antibody

to HSV was assayed by the method of Bradstreet andTaylor (2). Titers were converted to log2, and thegeometric mean titer was calculated.Lymphocyte cultures. Approximately 50 ml of

blood, of which 10 ml was allowed to clot, was obtainedfrom each individual, and the serum was separated.The remaining 40 ml was anticoagulated with heparin,layered onto Ficoll-Triosil (Pharmacia, Nyegaard),and centrifuged at 900 x g for 20 min (1). The cells atthe interface of Ficoll-Triosil and plasma were re-moved, washed twice in Hanks balanced salt solution,and suspended at 106/ml in medium 199. Cultureswere established in disposable tissue culture tubes(Falcon 3033) and consisted of 1 ml of lymphocytesuspension, 0.1 ml of serum, and 0.1 ml of either saline,

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antigen, or phytohemagglutinin (PHA). Sera werefresh autologous, except where stated otherwise. Ho-mologous sera had been stored at -20°C for up to 4weeks. Either two or three replicates of each culturewere used and were maintained at 370C in an atmos-phere of 5% C02 for 4 days.

For the last 4 h of culture, 1 ,uCi of [3H]thymidine(5 Ci/mmol) (TRA 120, Amersham) in 0.1 ml of me-dium was added to each culture. Cultures were ter-minated by filtration onto Whatman GFA filter paper,using a multiple automated sample harvester. Filterswere washed with saline and dried, and radioactivitywas assayed by scintillation counting. The meancounts per minute of replicate cultures stimulated byeach antigen, or PHA, was divided by that of theunstimulated control cultures; this value was recordedas the "stimulation index" (SI).

Antigens. (i) HSV. HSV-1 complement-fixing an-tigen was kindly supplied by C. M. P. Bradstreet,Public Health Laboratory Service, Colindale, London.A confluent monolayer of Vero tissue culture cellswas infected with a recent isolate of HSV-1 from apatient with RHL and incubated at 37°C until fullcytopathic effect was present. The infected cells wereharvested mechanically and centrifuged at 900 x g for10 min. The pellet was resuspended in peptone waterto 10% of the original volume and disrupted by soni-cation in an MSE ultrasonic disintegrator at 20 kc/s.The virus was inactivated by addition of 0.3% ,B-pro-piolactone, but no preservative was used. In one ex-perment only, a similar preparation of a genital isolateof HSV-2 was used.

(ii) PPD. Preservative-free purified protein deriva-tive (PPD) was obtained from the Ministry of Agri-culture, Fisheries and Food and was dissolved in saline.

(iii) Candida albican& A sample of saliva froman oral carrier of C. albicans was inoculated ontoSabouraud agar and incubated at 37°C until a con-fluent growth was obtained. The yeast cells were har-vested in the minimum possible volume of saline anddisrupted by vibration in a Mickle glass-bead disinte-grator. The suspension was centrifuged at 900 x g for20 min, and the supernatant was used.PHA. Reagent-grade PHA was obtained from Well-

come Reagents Ltd., and each vial was reconstitutedin 5 ml of distilled water.Each antigen was stored in 1-ml samples at -20°C

for up to 6 months before use, but PHA was freshlyreconstituted on each occasion. The optimal dilutionof each antigen and PHA was determined by compar-ison of lymphocyte responses of a number of subjectsto several dilutions of the preparation, as describedpreviously (23a).Macrophage migration inhibition. The indirect

test of Thor et al. (27) was used, as described previ-ously (29). The supernatant fluids from the lympho-cyte cultures were assayed for macrophage migrationinhibitory activity, with oil-induced guinea pig peri-toneal cells. Each supernatant was assayed with fourcapillary tubes of exudate cells, and the migrationindex was calculated by dividing the mean area ofmigration in antigen-stimulated supernatant by themean area of migration in unstimulated supernatantand multiplying by 100.

Statistical analy8is. Results were analyzed by the

t test for dependent or independent means, as appro-priate, or by analysis of linear regression.

RESULTSHSV antibody titer. Control subjects with

HSV antibody titers greater than 1:4 were des-ignated seropositive (11 subjects) and the re-mainder were seronegative (8 subjects).

All patients had an HSV antibody titer of 1:4or greater, and no difference was found betweenthe titer when a lesion was present (geometricmean titer ± standard error = 4.31 ± 0.25) andthat after the lesion had resolved (4.64 ± 0.27).Lymphocyte stimulation. Unstimulated

lymphocytes of patients showed no variation inthymidine incorporation with time since onsetof a lesion, and no difference was seen betweencontrol subjects and patients.HSV. The highest mean response to the HSV

antigen was found in lymphocytes of patientswith a recurrent herpetic lesion of up to 3 days'duration, and at each subsequent time of exam-ination the mean response had declined (Fig.1). The SI to HSV when a lesion had beenpresent for up to 3 days was significantly greaterthan the SI 28 days after the onset of lesions (P< 0.05). The SI of the patients was significantlygreater than that of the seropositive controlswhen less than 3 days (P < 0.001) or 14 to 21days (P < 0.05) had elapsed since the onset ofthe lesion. After 28 days had elapsed, the meanSI in response to HSV was still higher than thatof the seropositive controls (Fig. 1), but thedifference no longer reached the 5% level ofsignificance.The increase in the lymphocyte response to

HSV at the time of a herpetic lesion was repeat-

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FIG. 1. Stimulation indexes to HSV-1 of lympho-cytes from controls and patients at various timesafter onset of a recurrent herpetic infection. The SIis the ratio of counts per minute in HSV-stimulatedcultures to countsper minute in unstimulated culturesfrom the same individual. Mean values + standarderror are indicated for each group of subjects.

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132 SHILLITOE, WILTON, AND LEHNER

edly observed during a sequential study of onepatient who experienced 12 episodes of RHLduring a period of 10 months (Fig. 2). When aherpetic lesion was present, the SI was invaria-bly greater than before or after the onset of alesion. The maximum number ofherpetic lesionspresent at the same time was six, and this coin-cided with the maximum SI in response to HSVof 49.0.The lymphocyte response to HSV at the time

of a herpetic lesion was related to the timeelapsed since the previous lesion in 16 patients.The shorter the time elapsed since the previouslesion, the higher the SI, although the correla-tion did not reach the 5% level of significance(Fig. 3). However, if only values for up to 90days since the previous lesion were considered,the correlation did reach the 5% level of signifi-cance (r = -0.65, n = 10,P< 0.05). No significantrelationship was observed between the lympho-cyte response to HSV at the time of a herpeticlesion and the time that elapsed before the nextlesion developed (r = -0.41, n = 9).The specificity of the lymphocyte response

for HSV-1 was assessed by comparing the re-sponses between HSV-1 and HSV-2 of lympho-cytes from 15 patients prone to RHL. No signif-icant difference was seen between the mean SIto HSV-1 (12.4 ± 2.6) and HSV-2 (14.3 ± 3.5),and the correlation between the responses toeach antigen was highly significant (P < 0.0001;Fig. 4).PPD. No difference was seen between the

lymphocyte responses to PPD of patients with

50

40

30j

20

18

16

X 14

- 12z0

I10

a herpetic lesion of up to 7 days' duration (SI =11.3 ± 4.2) and patients in whom more than 14days had elapsed (SI = 11.9 ± 7.8). The re-sponses ofthe seronegative and seropositive con-trols were combined, showing a mean SI of 6.6± 2.0, which was not significantly different fromeither of the patient groups.

4c

30

25

20

1 5

x

100

c3 5

r o

N= 16r= - 0.47

0

0

0

0

0

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S

00

GU nU oU OU *JuuDays since previous lesion

FIG. 3. Relationship between response of lympho-cytes to HSV-1 at the time of a herpetic lesion andthe interval since the previous lesion in 16 patients.

Feb. 1975 Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Jan. 1976

FIG. 2. Relationship between presence ofherpetic lesions and response to HSV-1 in one subject.

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430

28

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c 18

lv 161glo

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0 10

a 8

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r = 0.87N = 15p < 0. 0001

2 4 6 8 10u 12 14 16 10 0z 22 24 26 28 X30STIMULATION INDEX: HSV-1

FIG. 4. Correlation between lymphocyte responses to HSV-1 and HSV-2 of 15 patients prone to RHL.

C. albicanu Patients with a herpetic lesionshowed a mean SI to C. albicans of 8.3 ± 1.7,which increased slightly to 10.1 ± 3.5 after morethan 14 days had elapsed. The responses of theseronegative and seropositive controls werecombined, showing a mean SI of 5.9 ± 1.1, whichwas not significantly different from either of thepatient groups.PHA. No difference was seen between the

lymphocyte responses to PHA of patients witha herpetic lesion of up to 7 days' duration (SI =60 ± 11.7) and patients in whom more than 14days had elapsed (SI = 63.5 ± 13.8). The re-sponses ofthe seronegative and seropositive con-trols were combined, showing a mean SI of 87+ 12.9, which was not significantly different fromeither of the patient groups.

Unlike with HSV, no relationship was ob-served between the lymphocyte responses of thepatients and time elapsed since the previousherpetic lesion if either C. albicans (r = -0.37, n= 6) or PHA (r = -0.56, n = 6) was used as thestimulant. However, fewer patients were testedthan with HSV.

Effect of substituted sera. Lymphocytes ofsix seronegative control subjects were culturedin the presence of serum from seropositive sub-jects. A total of 11 seropositive sera was used; 3were from control subjects, 5 were from patientswho had developed a recurrent herpetic lesionwithin the previous 7 days (acute phase), and 3were from patients whose previous lesion haddeveloped more than 14 days previously (nona-cute phase). The SI to HSV of lymphocytes

from seronegative controls was not increased inthe presence of seropositive as compared to ser-onegative (autologous) serum (Table 1).Lymphocytes of eight seropositive subjects

were cultured in the presence of serum fromseronegative controls. The seropositive subjectswere three acute-phase patients, four nonacute-phase patients, and one control. The SI in re-sponse to HSV was not significantly decreasedin the presence of seronegative as comparedwith autologous serum (Table 1).Lymphocytes of 11 acute-phase patients were

cultured in the presence of nonacute-phase sera,which had been collected previously. The non-acute-phase sera were autologous in four casesand homologous in six cases, but since no differ-ences were seen between them, the results werecombined. The SI in response to HSV in thepresence of autologous fresh acute-phase serumwas 17.6 ± 3.5, and in the presence of nonacute-phase sera it was 15.6 ± 4.3. The decrease wasnot significant at the 5% level.Lymphocytes of six nonacute-phase patients

were cultured in the presence of three autolo-gous acute-phase sera, two autologous and ho-mologous acute-phase sera, or one homologousacute-phase serum. The mean counts per minuteof unstimulated lymphocytes was slightly de-pressed by acute-phase serum, whereas those ofHSV- and C. albicans-stimulated lymphocyteswere slightly increased, when compared withcultures in the presence of autologous nonacute-phase serum (Table 2). None of these changesapproached the 5% level of significance. How-

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134 SHILLITOE, WILTON, AND LEHNER

ever, when SIs to HSV were calculated, an in-crease was seen in the presence of acute-phaseserum (P < 0.05), and a corresponding increasewas also found with C. albicans (P < 0.01; Table2).Macrophage migration inhibition. (i)

HSV. Supematants of HSV-stimulated lympho-cytes of seropositive and seronegative controlsshowed macrophage migration indexes that didnot differ significantly from each other. Obser-vations were made in eight patients, both whena recurrent herpetic lesion was present and afterits resolution (Fig. 5). The mean time betweenthe observations was 25 days. An increase inmigration inhibition was observed in six cases;the maximum change in migration index was-77. A decrease in migration inhibition was ob-served in two cases; the maximum change inmigration index was +8. The mean increase inmigration inhibition between the two observa-tions was significant at the 5% level. At the timeof the second observation the mean migrationindex was 59 + 7, which differed significantlyfrom that of the seropositive controls (mean =87 ± 6, P < 0.01).

(ii) PPD. No difference was seen between themigration inhibition response to PPD of lym-phocytes from patients with a herpetic lesion ofup to 7 days' duration (migration index = 83 ±9), patients in whom more than 14 days hadelapsed (90 ± 11), or control subjects (94 + 5.5).

(iii) C. albicans. No difference was seen be-

TABLE 1. Effect of substituted seropositive orseronegative sera on the response to HSV oflymphocytes from seronegative or seropositive

donorsLymphocyte donor Serum donor SIa

Seronegative Seronegative 3.3 ± 0.8Seropositive 2.7 ± 0.6

Seropositive Seropositive 14.7 ± 5.1Seronegative 12.3 ± 3.0

a Ratio of counts per minute in HSV-stimulatedcultures to unstimulated cultures containing the sameserum. Mean ± standard error.

tween the migration inhibition response to C.albicans of lymphocytes from patients with aherpetic lesion of up to 7 days' duration (migra-tion index = 99 + 13) or patients in whom more

than 14 days had elapsed (103 ± 13.5). Controlsubjects showed rather greater migration inhi-bition (mean migration index = 70 ± 8.5), butthe difference did not reach the 5% level ofsignificance.

DISCUSSIONPrimary herpetic infections result in the de-

velopment of HSV antibody (3) and the main-tenance of the virus in the local sensory ganglionin a latent form (26). A minority of infectedindividuals later suffer recurrent infections, de-spite the presence of circulating antibody, andthe present study investigated the role of cell-mediated immunity in the pathogenesis of re-

currences. The highest mean SI to HSV was

seen in lymphocytes from patients with herpeticlesions of less than 3 days' duration, and the

<30

40

50

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x 70

80z

p 90

2 100

110

120

130

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1LSeroneptive Seroposit.ve

C ontrols< 7d > 14dPatients: time sinee onset of lesion

FIG. 5. Migration indexes to HSV-1 oflymphocytesof controls and paired samples of patients with re-

current HSV infection. The migration index is theratio of area of migration of guinea pig peritonealexudate cells in thepresence ofsupernatants of virus-stimulated to that in the presence of supernatants ofunstimulated lymphocytes from the same individual.Mean values standard error are indicated for eachgroup of subjects.

TABLE 2. Effect of substituted sera from acute- or nonacute-phase patients on responses of lymphocytesfrom nonacute-phase patients

cpm SIaSerum donorSerumdnsimlaedHSnsimlaed C. albicans stimu- C. albicansUnstimnulated HSV stimulated lated HSV stimulated stimulated

Nonacute phase 2,312 ± 662b 13,825 ± 4,187 11,266 + 3,216 6.5 ± 0.9 4.0 ± 1.1Acute phase 1,531 ± 207 14,667 ± 2,406 16,811 ± 3,250 10.6 ± 1.4' 9.3 ± 1.2d

a Ratio of counts per minute in antigen stimulated to unstimulated cultures containing the same serum.I Mean ± standard error.c Increase in stimulation index in presence of acute-phase sera significant at P < 0.05 level.d Increase in stimulation index in presence of acute-phase sera significant at P < 0.01 level.

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response declined after resolution of the lesion(Fig. 1). A similar fall in response was found byRosenberg et al. (21) but not by other workers(17, 25). Patients with a lesion showed a higherresponse to HSV if only a short time had elapsedsince the previous lesion, but the correlationreached the 5% level of significance only whenpatients were excluded if their previous lesionhad occurred more than 90 days earlier. Thismight indicate that the SI in response to HSVdeclines linearly with time for about 90 daysand then remains constant at a low level. Theincreased response at the time of a lesion wasevidently associated with a period of suscepti-bility to a recurrence, and not with resistanceto infection: the SI was probably increased byantigenic stimulation from virus replicating inthe lesion.

It was previously suggested that recurrentherpetic infections could result from inability ofthe patients' lymphocytes to release migrationinhibitory factor after stimulation by HSV (29).In the present study, supernatants of HSV-stim-ulated lymphocytes from seropositive controlsand of patients with a herpetic lesion did inhibitthe migration of guinea pig peritoneal macro-phages, but this response increased significantlyduring recovery from infection (Fig. 5). Sequen-tial observations were made in only eight sub-jects, and the results therefore require confir-mation in a larger series. Nevertheless, the as-sociation between an increase in the responseand recovery from infection is consistent withthis mechanism being protective against infec-tion, unlike the SI, which fell simultaneously.The mechanism by which release of migration

inhibitory factor by HSV-stimulated lympho-cytes could be protective against recurrent HSVinfection is uncertain. However, as well as inhi-bition of migration, soluble mediators of cell-mediated immunity potentiate the bactericidal(16) and viricidal functions of macrophages, andin animals macrophages are essential in protec-tion against HSV infection (11). The solublemediators of cell-mediated immunity are usuallyreleased simultaneously (18), and a lack of mi-gration inhibitory factor might be associatedwith a lack of other mediators. Although inter-feron production is within the normal range, thelower the concentration, the shorter the timebefore the next herpetic lesion (9, 17). However,production of chemotactic factor and lympho-toxin seems to be normal (21). A cyclical pattemin the release of a soluble mediator of cell-me-diated immunity is supported by O'Reilly andLopez (16a) and may account for the negativeresults in those investigations in which the testswere carried out in the absence of lesions (23).An altemative interpretation of the cyclical

change in the migration inhibition response isthat viral infection depresses some lymphocytefunctions. However, immunosuppression by vi-ruses in vivo results in loss of lymphocyte re-sponses to unrelated antigens and mitogens (5,10, 28), which was not detected in the presentstudy when PPD, C. albicans, or PHA was usedas the control. It would seem possible that aninsufficient migration inhibition response istherefore a predisposing cause rather than aresult of infection, although a lesion may onlydevelop when this coincides with local factors,allowing derepression of the viral genome (15)and spontaneous shedding of viral particles (4).The possibility that the response of lympho-

cytes to HSV might be modulated by serumfactors was not supported by the results of thisinvestigation. Lymphocytes from patients proneto RHL responded equally well to HSV by thy-midine incorporation whether cultured in thepresence of autologous or homologous sero-negative serum. The increased lymphocyte re-sponse from a donor with a lesion was main-tained in the presence of serum from a donorwithout a lesion. The lymphocyte response wasnot increased when the cells from a donor with-out a lesion were cultured in the presence ofserum from a donor with lesions. However, thelatter sera caused a slight decrease in the [3H]-thymidine uptake of unstimulated lymphocytesand a slight increase in the uptake in responseto HSV or C. albicans, as compared with thatin the presence of autologous fresh serum. As aresult, the SIs to both antigens were raised, andthe increase reached the 5% level of significancein the case of C. albicans. However, since thechange in SI was seen with two unrelated anti-gens, and was largely due to a fall in backgrounduptake of thymidine, it was considered to beimmunologically nonspecific. These results sug-gest that the varying responses of lymphocytesfrom patients prone to RHL are entirely cellmediated and are not related to serum factors.Although a slight increase in the response oflymphocytes in the presence of seronegative ascompared with seropositive serum was recentlyreported (9), this was not statistically significant.Hyperimmune serum can inhibit the re-

sponses of rabbit spleen lymphocytes to cell-bound, but not cell-free, HSV (6, 19, 20), al-though in humans it is probable that the anti-body titer is insufficient to cause lymphocytesuppression. The immunoglobulin class of theantibody may also be relevant, since inhibitionof the response to rubella of rabbit lymphocytesby serum is due to immunoglobulin M antibodyalone (14). The lack of any influence of differentsera on the lymphocyte responses to HSV seenin the present study is consistent with both the

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lack of immunoglobulin M antibody to HSV inserum ofpatients with RHL (8) and the constanttiter ofwhole serum antibody to HSV, regardlessof the presence or absence of a lesion; this wasseen in the present and in past investigations(4, 13, 17).A very significant correlation was observed

between lymphocyte responses to HSV-1 andHSV-2 (Fig. 4), suggesting that lymphocytes arestimulated by an antigen common to both HSVtypes. This is in contrast to the finding thatrabbits immunized with one HSV type havelymphocytes sensitized to both types but astronger response to the immunizing type (12,22). Infants with neonatal HSV-2 infection de-velop a stronger lymphocyte response to HSV-2 than to HSV-1 (24). The adult patients in thepresent study were prone to RHL, which isusually due to HSV-1, but it cannot be excludedthat some had had an HSV-2 infection in thepast.The sequential observations during the pres-

ent study have shown that cyclical changes oc-cur in the specific cell-mediated immune re-sponse to HSV related to the presence of aherpetic lesion. It is suggested that a periodicdefect in the macrophage migration inhibitionresponse to the virus allows the infection todevelop, and that the increased lymphoprolifer-ative response then results from antigenic stim-ulation by the virus replicating in the lesion.Recent observations suggest that both the mac-rophage inhibition and lymphoproliferative re-sponses in this system are functions of the Tlymphocyte (23a).

ACKNOWLEDGMENTSThe technical assistance of J. Blackburn and J. Walton is

gratefully acknowledged.This work was carried out under a grant from the Cancer

Research Campaign.

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3. Dascomb, H. E., C. V. Adair, and N. Rogers. 1955.Serologic investigations of herpes simplex virus infec-tions. J. Lab. Clin. Med. 46: 1-11.

4. Douglas, R. G., and R. B. Couch. 1970. A prospectivestudy of chronic herpes simplex virus infection andrecurrent herpes labialis in humans. J. Immunol.104:289-295.

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7. Gange, R. W., A. D. Bats. J. R. Park, C. M. P. Brad-street, and E. L. Rhodes. 1975. Cellular immunityand circulating antibody to herpes simplex virus insubjects with recurrent herpes simplex lesions and con-trols as measured by the mixed leucocyte migrationinhibition test and complement fixation. Br. J. Derma-tol. 93:539-544.

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13. Kurtz, J. B. 1974. Specific IgG and IgM antibody re-sponses in herpes-simplex-virus infections. J. Med. Mi-crobiol. 7:333-341.

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15. Lehner, T., J. M. A. Wilton, and E. J. Shillitoe. 1975.Immunologic basis for latency, recurrences and putativeoncogenicity of herpes simplex virus. Lancet ii:60-62.

16. Nathan, C. F., H. G. Remold, and J. R. David. 1973.Characterisation of a lymphocyte factor which altersmacrophage functions. J. Exp. Med. 137:275-290.

16a.O'Reilly, R. J., A. Chibbaro, E. Anger, and C. Lopez.1977. Cell-mediated immune responses in patients withrecurring herpes simplex infections. II. Infection asso-ciated deficiency of lymphokine production in patientswith recurrent herpes progenitalis. J. Immunol.118:1095-1102.

17. Rasmussen, L. E., G. W. Jordan, D. A. Stevens, andT. C. Merigan. 1974. Lymphocyte interferon produc-tion and transformation after herpes simplex infectionsin humans. J. Immunol. 112:728-736.

18. Rocklin, R. E. 1975. Inhibition of cell migration as acorrelate of cell mediated immunity, p. 111-126. In G.Vyas et al. (ed.), Laboratory diagnosis of immunologicdisorders. Grune and Stratton, New York.

19. Rosenberg, G. L., P. A. Farber, and A. L. Notkins.1972. In vitro stimulation of sensitised lymphocytes byherpes simplex virus and vaccinia virus. Proc. Natl.Acad. Sci. U.S.A. 69:756-760.

20. Rosenberg, G. L., and A. L. Notkins. 1974. Inductionof cellular immunity to herpes simplex virus: relation-ship to the humoral immune response. J. Immunol.112:1019-1025.

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