CD8 + Cell Anti-HIV Activity: Nonlytic Suppression of Virus Replication

AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 8, Number 6, 1992Mary Ann Liebert, Inc., Publishers

CD8+ Cell Anti-HIV Activity: Nonlytic Suppressionof Virus Replication

CARL MACKEWICZ AND JAY A. LEVY

INTRODUCTION

Acquired immunodeficiency syndrome (AIDS) is charac¬terized by the slow deterioration of the immune system,

believed to result from the loss of CD4+ lymphocytes, the majortarget cell of the virus. This effect is most dramatically demon¬strated by the development of a variety of opportunistic infec¬tions and certain types of cancers in the host. ' The outcome ofHIV. infection is conceivably influenced by many factors,including (1) characteristics of the virus, such as host range,replicative ability, cytopathicity, and its ability to mutate; (2) theimmune response, both humoral and cellular; and (3) the geneticmakeup of the individual that may influence the susceptibility ofcells to virus infection and the extent and specificity of theimmune reaction to the virus.

Of particular interest is how the virus persists in the hostdespite apparently normal immune responses against it. Themajority of HIV-infected individuals mount, at least during theearly stages of infection, an antibody response that is capable ofneutralizing the virus,2'3 as well as mediating antibody-depen¬dent cellular cytotoxicity (ADCC)4"6 and complement-depen¬dent lysis of infected cells.7·8 Most of the classical cell-mediatedmechanisms have also been found present during HIV infection,including natural killer (NK) cell activity,9·10 ADCC mediatedby macrophages and NK cells,4"6 and CD8+ T-cell effectorfunctions. " · '2 In regard to the latter, antigen-specific cytotoxic lymphocytes (CTL), normally important in combating viralinfections, are readily detectable in HIV infection. They havebeen demonstrated in various tissues and are capable of lysingtarget cells expressing HIV proteins including env, gag, pol,nef, and ví/.11·13"15

HIV-infected individuals also possess CD8+ lymphocytesthat can suppress HIV replication in vitro by a nonlytic mecha¬nism. This phenomenon was first shown by Walkeret al.,12 andsubsequently confirmed by others.16'17 Unfortunately, the rela¬tive contributions of these various immune responses to themaintenance of the clinically healthy state during HIV infectionis unclear. Our laboratory has been actively characterizing thenonlytic CD8+ cell anti-HIV activity in attempts to understandits importance to the host during HIV infection. Its value in

directing therapeutic approaches is being considered. This arti¬cle reviews our current understanding of this type of naturalimmune antiviral response.

ANTI-HIV SUPPRESSIVE ACTIVITYMEDIATED BY CD8+ CELLS IN VITRO

Antiviral activity against naturally infected CD4+ cellsSoon after the identification of HIV, it was observed that

cultured peripheral blood mononuclear cells (PMC) from >50%of seropositive healthy individuals did not yield infectious virus.Walker et al.12 demonstrated that CD8 + cells were responsiblefor this lack of HIV production in culture. Removal of CD8 +

cells from the PMC by specific immunoselection prior to theirculture with phytohemagglutinin (PHA) resulted consistently inHIV replication as detected by elevated levels of reverse tran¬

scriptase (RT) activity in the culture fluid. However, if CD16+(Leul 1) cells, primarily NK cells, were removed from the PMC,no virus replication was observed.

Subsequent reconstitution of the CD8+ cell-depleted popula¬tion (prior to culture) with graded numbers of CD8+ cellssubstantiated that this antiviral activity was mediated by CD8+cells and indicated that it was dose dependent. In some cases,CD8+/CD4+ (effector/target) cell ratios as low as 1/5 were

found to prevent HIV replication. Moreover, when isolatedCD8+ cells were added to cultures of autologous CD8+ cell-depleted PMC actively producing HIV, virus replication was

suppressed within 7-10 days. This finding indicated that theCD8+ cells were not merely blocking the induction of HIVreplication (i.e., from a proviral state), but were capable ofsuppressing active replication. The CD8+ cells cultured sepa¬rately could maintain this antiviral activity for at least threeweeks. Conversely, if the CD8+ cells were removed 2-3 weekspost-PHA stimulation of cultured HIV-infected PMC, highlevels of HIV replication could subsequently be detected withinthe CD8+ cell-depleted fraction. Importantly, in these mixedcultures, CD4+ cell percentages remained relatively unchangedduring HIV suppression.12 In addition, recent experiments haveshown that CD8+ cells do not inhibit the expression of the

Department of Medicine, Cancer Research Institute, University of California, School of Medicine, San Francisco, CA 94143-0128.

1039

1040 MACKEWICZ AND LEVY

Table 1. Activation Markers Expressed on NaturallyInfected CD4+ Cells Exposed in Culture to

CD8+ Cells3

RTCD38 CD25 HLA-DR Activity0

Subject #1CD4CD4/CD8C

Subject #2CD4CD4/CD8

96100

9599

5850

5250

2425

1816

4692.3

13491.5

a Coexpression of the listed activation marker on CD4+ cellswas evaluated by dual-color flow cytometry on day 9 of culture,just prior to detectable HIV release in the endogenous assay.

b Peak reverse transcriptase (RT) activity in the culturefluid—usually on day 12.

c CD8 + cells were mixed with the CD4+ cells at a ratio of 1:4on day 0.

activation markers CD25, CD38, and HLA-DR on the CD4+cells (Table 1). These observations suggest that the CD8+ cellssuppressing HIV replication are not inhibiting CD4+ cell acti¬vation, nor are they killing infected CD4+ cells to any signifi¬cant degree.

Cytometric analysis of the purified CD8+ cell populations hasshown that 97-99% of the cells stain positive for CD3, a

T-cell-specific marker. Furthermore, when autologous purifiedmacrophages or PMC depleted of both CD4+ and CD8+ cellswere added to naturally infected CD4+ cell cultures, suppres¬sion of HIV replication did not result.18 Taken together, thesestudies indicated that HIV-infected individuals possess CD8+ cells in their peripheral blood capable of inhibiting in vitroreplication of HIV in CD4+ lymphocytes, without adverselyaffecting them.

In some individuals even very small numbers of CD8+ cellscan inhibit HIV replication when added back to CD8-depletedPMC.19 This result may explain why certain CD8+ cell-de¬pleted cultures did not yieldvirus; some contaminating antiviralCD8+ cells were still present.19 It also suggested, as was

subsequently shown19 and confirmed,20 that culture of purifiedCD4+ cells from PMC of HIV seropositive individuals wouldprovide the best method for in vitro isolation of virus. Use ofpurified naturally infected CD4+ cells as targets has now

become the basis for what is called the "endogenous" assay,referring to the replication of virus naturally infecting the cell(Fig. 1). In this assay the infected CD4+ cells from an HIV-infected individual are cultured alone or together with autolo-gousCD8+ cellsatarangeofCD8/CD4ratios. Virus replicationis then monitored every three days by measuring RT activity inthe culture fluids. This assay has proved to be very useful in theevaluation of the extent of this type of CD8+ cell-mediatedanti-HIV activity in infected individuals.

Similar activity mediated by CD8+ cells has also beenobserved in studies with simian immunodeficiency virus (SIV)infected rhesus monkeys,16·21 and in HIV-1-infected chimpan¬zees.22 It can be detected as early as 2-4 weeks postinfection inmonkeys.16 In both the monkey and chimpanzee systems,

Patient PMC° o O

o O °

ImmunomagneticBead Separation

o°ooo oo o

CD8+ Cells(97-99% pure)

°o OOqO


mix at selectedratios

culture alone

3 Days vith PHA

Pass every 3 days in IL-2 supplemented medium lacking PHAand monitor HIV replication by reverse transcriptase activityin the culture fluid.

FIG. 1. Endogenous assay for measuring CD8+ cell suppres¬sion of HIV replication. CD4+ and CD8+ cells are isolated fromperipheral blood mononuclear cells (PBMC) of HIV-infectedindividuals by using immunomagnetic beads specific for therespective cell marker.25 The CD4+ and CD8+ cells are thenmixed at selected ratios and cultured for 3 days in the presence ofPHA (3 µg/mI) in IL-2-supplemented medium. Every 3 daysthereafter, the cultures are passed with fresh medium lackingPHA and the culture supernatants are assayed for reverse

transcriptase activity.63

depletion of CD8+ cells from PMC obtained from infectedanimals allowed for virus replication from the otherwise nonper¬missive cells. Furthermore, in both animal models, the CD8 +

cell-mediated suppressing activity was dose dependent, and didnot eliminate the infected CD4+ cells in culture. Thus, thisnoncytotoxic antiviral response occurs in nonhuman primates as

well.

Antiviral activity against acutely infected CD4+ cellsBecause many severely ill HIV-infected patients have too few

CD4+ cells to examine CD8+ cell anti-HIV activity with theendogenous assay, an alternative procedure using exogenousvirus infection was developed (Fig. 2). By this procedure,CD4+ cells obtained from uninfected donors are stimulated withPHA and infected with HIV. Mitogen-activated CD8+ cells are

then mixed with the CD4+ cells and the cultures are monitoredfor virus replication. Referred to as the "acute" assay herein, thisprocedure permits examination ofCD8+ cell responses against a

variety of virus isolates with different biologic properties. Using

NONLYTIC SUPPRESSION OF VIRUS REPLICATION 1041

Patient PMCSeronegative

Donor PMC° o O

o o

I m m uno magnetiBead Separati

eticion

ImmunomagneticI Bead Separation

o°ooo oo o

CD8+ Cells(97-999B pure)


Infect vit hHIY for Ihr

\ / Imix at selected

ratiosculture alone

1 1Pass every 3 days in IL-2 supplemented medium lacking PHAand monitor HIV replication by Reverse Transcriptase activityin the culture fluid.

FIG. 2. Acute assasy for measuring CD4+ cell antiviral activity. The CD8+ effector cells are obtained from PHA-stimulatedperipheral blood mononucler cells (PMC) of HIV-infected individuals using CD8-specific immunomagnetic beads. The infectedCD4+ targets are obtained by isolating CD4+ cells from PMC of HIV seronegative donors using CD4-specific immunomagneticbeads. The CD4+ cells are stimulated with PHA (3 µg/ml) for 3 days, washed, and then infected with virus at a known input dosefor 1 hour. After washing out nonbound virus, the infected CD4+ cells are mixed with effector CD8+ cells at selected ratios andcultured in IL-2-supplemented medium.23-25 Every 3 days the cultures are passed with fresh medium and the culture supernatantsare assayed for reverse transcriptase activity.63

this assay, Walker et al.23 found that CD8+ cells from HIV-1-infected individuals were able to suppress the replication of a

variety of different HIV-1 and HIV-2 strains as well as SIV.Similar observations have been made with CD8+ cells frominfected chimpanzees.22 Thus, both high replicating, highlycytopathic viruses, as well as those with less replicating capac¬ity, could be suppressed by CD8+ cells from infected individu¬

als. Nevertheless, slower replicating strains appear more sensi¬tive in mosteases.

Analogous to the anti-HIV activity exhibited by the CD8 +

cells in the endogenous assay, cell killing of the infected CD4 +

cells does not appear to be responsible for the inhibition of HIVreplication detected in the acute assay24 (see below). Studieswith monozygotic twins have indicated that the ability of the


CD8+ cells to suppress HIV replication is not the consequenceof an allogeneic response to the heterologous CD4+ cell tar¬

gets.24 CD8+ cells from the infected twin suppress HIV replica¬tion in acutely infected CD4+ cells from the HLA-identicaluninfected twin just as well as they suppress replication inheterologous CD4+ cells. Moreover, CD8+ cells from unin¬fected donors do not suppress HIV replication in acutely infectedheterologous CD4+ cells.23

Aside from the usefulness of the acute assay for studyinginfected individuals with very low CD4+ cell numbers, thisprocedure also has the following advantages: (1) the activityagainst a variety of HIV strains can be tested, in addition to theautologous strain; (2) the number of CD4+ cells infected (i.e.,virus load) can be controlled by the known TCID50 dose of theinfectious virus; (3) CD8+ cell response among different indi¬viduals can be evaluated using the same virus strain and the same

CD4+ cells.

<

4.0

2.0

1.0

0.5

ö 0.25 0.1

0.05

Endogenous Assay Acute Assay

CLINICAL RELEVANCE

Extent of CD8Aclinical state

cell-mediated activity correlates with

Having observed this in vitro phenomenon of CD8+ cellsuppression of HIV replication and some of its characteristics,its clinical relevance became an important question. Is it presentin vivo, and if so, what role does this immune response play inprotecting the host from disease? Studies by Walker et al.19revealed that some individuals exhibited a higher degree ofCD8+ cell anti-HIV activity than others, and noted that whilecultures of unfractionated PMC from most asymptomatic HIV-infected individuals do not replicate HIV, analogous culturesfrom some AIDS patients do. This finding suggested that theseAIDS patients have decreased CD8+ cell-mediated anti-HIVactivity.

To address these questions, a point prevalence study examin¬ing 42 different subjects at various clinical stages of HIVinfection, assessed the relative degree of anti-HIV activityexhibited by their CD8+ lymphocytes.25 The results of thisstudy indicated that a direct, and highly significant correlationexists between the clinical status and the number of CD8+ cellsrequired to inhibit HIV replication. Asymptomatic individualsexhibited the highest degree of anti-HIV activity (1/20, CD8/CD4 ratio), whereas AIDS patients showed the least (4/1 ratio insome cases). The relative degree of CD8+ cell-mediated anti-HIV activity also directly correlated with the subject's periph¬eral blood CD4+ cell count, but not CD8+ cell count.

Conceivably, virus load and virus variation could have influ¬enced the degree to which CD8+ cells were able to inhibit HIVreplication in these studies. This possibility was examined bytwo approaches. The first relied on the ability of CD8+ cells toinhibit HIV replication in naturally infected heterologous CD4+cells (the endogenous assay). CD8+ cells that exhibited rela¬tively weak anti-HIV activity against naturally infected autolo¬gous CD4+ cells (i.e., CD8+ cells from ARC or AIDS patients)were found to have weak activity against naturally infectedheterologous CD4+ cells from asymptomatic individuals. Con¬versely, CD8+ cells with relatively strong antiviral activity

SUBJECT

FIG. 3. Extent of CD8 + cell anti-HIV-1 activity as measuredby the endogenous and the acute virus infection assays. Openbars represent results obtained when anti-HIV-1 activity by thesubject's CD8+ cells was titrated against autologous naturallyinfected CD4+ cells (endogenous virus assay) with a range ofeffector/target (E/T) ratios. Dark bars represent results obtainedwhen the CD8+ lymphocytes from the same individual weretitrated against CD4+ lymphocytes acutely infected with HIV-1SF33 (acute virus infection assay). The extent of antiviralactivity exhibited is represented as the lowest effector/target(E/T) ratio yielding ^90% reduction of reverse transcriptaseactivity in the culture fluid compared to CD4+ cells culturedalone. (Reproduced from the Journal of Clinical Investigation,1991, 1462-1466 by copyright permission of the AmericanSociety for Clinical Investigations.)

against infected autologous CD4+ cells (i.e., from asympto¬matic individuals) exhibited strong activity against infectedheterologous CD4+ cells from AIDS patients. Thus, the extentof the CD8+ cell anti-HIV response did not depend on theparticular CD4+ cells used nor the virus they harbored, butseemed to be dependent solely on the intrinsic activity of theCD8+ cells.

The second approach utilized the acute assay, thereby virtu¬ally eliminating variations due to different virus strains, or virusloads, as well as differences among CD4+ cells. In agreementwith results from the endogenous assay, these studies indicatedthat CD8+ cells from asymptomatic individuals exhibited thehighest degree of anti-HIV activity, and those from AIDspatients exhibited the least.25 As can be seen in Figure 3, higherinitial effector/target ratios were usually necessary to controlvirus replication in acutely infected CD4+ cells as opposed to

naturally infected cells. This difference between the two assayscould be a result of a greater virus load and/or the replicationpotential of the highly cytopathic HIV strain used to acutelyinfect the cells. A limited degree of this CD8+ cell anti-HIVactivity in AIDS patients has also been reported by others.17-26Taken together, the above studies support the conclusion that the


extent of this anti-HIV response reflects the intrinsic activity ofthe CD8+ cells and strongly suggest that this anti-HIV activityhas clinical relevance.

But the question still remained, how important is this immunefunction to the maintenance of a clinically healthy state. Doesloss of this activity in HIV-infected individuals lead to disease?The latter question has been addressed in part by longitudinalstudies that assess the degree of CD8+ cell anti-HIV activityexhibited by individuals overtime. To date, no one in our cohortof subjects has clinically progressed, and in many, the degree ofantiviral activity has remained stable for almost two years.However, there are some individuals whose CD8+ cell activityhas been decreasing. We suspect that these individuals will soon

progress to disease. In a limited study evaluating cryopreservedsamples from five HIV-infected individuals over a 5-6-yearperiod, further evidence suggesting relevance of this antiviralactivity to the clinical state was obtained.27 Subjects whoseCD8+ cell anti-HIV activity against acutely infected CD4+targets decreased over time had a corresponding progression todisease. In these studies, the degree of antiviral activity seemedto be less dependent on absolute CD4+ cell numbers than theclinical state itself.

Finally, Kannagi et al.26 found that superinfection of PMC ofhealthy infected individuals with a laboratory strain of HIV-1did not lead to HIV replication. However, if the CD8+ cellswere removed prior to virus inoculation, replication of both theexogenously added HIV-1 strain as well as the endogenousautologous strain, was observed. Whether HIV-1 infection ofthe PMC occurred and was blocked after entry was not deter¬mined, but these studies do suggest a mechanism (i.e., CD8+cell activity) which protects individuals from superinfection byanother viral strain. The antiviral CD8+ cells might in some

cases establish a resistant state in the host preventing furthervirus infection and spread. Thus far, very few reports haveindicated more than one HIV-1 or HIV-2 strain in the same

individual.28,29

Antiviral activity of nonmitogen-stimulated CD8+ cells

Further evidence suggesting that this activity can act in vivocame from studies using the patients' CD8+ cells without priorin vitro stimulation. In all of the aformentioned studies CD8 +

cell anti-HIV activity reflected by suppression of HIV replica¬tion in target CD4+ lymphocytes was measured using CD8+cells exposed in culture to PHA, ConA, or anti-CD3-coatedimmunomagnetic beads. These reagents are capable of elicitinganti-HIV activity from patients' CD8+ cells, whether hu¬man1216·26 or nonhuman primates.21·22 But if this activity is tooccur in vivo, exogenous stimulation should not be necessary todemonstrate this CD8+ cell antiviral response in vitro. In fact, inmost HIV-infected individuals a large percentage of the CD8 +

cells exhibit an activated phenotype27'30-33 (see below), sug¬gesting that they are already activated in vivo.

In examining this parameter, freshly isolated (nonexoge-nously stimulated) CD8+ cells from two of three subjects were

found to inhibit HIV replication in acutely infected CD4+targets to nearly identical levels as did their respective PHA-stimulated CD8+ cells.27 Fresh CD8+ cells from the thirdsubject exhibited substantial anti-HIV activity, but fewer PHA-

stimulated CD8+ cells were required to inhibit HIV replicationin culture to the same extent as that needed for fresh CD8+ cells.Thus, we conclude that anti-HIV activity exhibited by CD8+cells in vitro is not dependent on exogenous stimulation.

Association of CD8+ cell anti-HIV activity withspecific subsets

The observation that this CD8+ cell anti-HIV activity pro¬gressively declines in infected individuals raised the question ofthe cause of its loss over time. CD8+ cell numbers generallyincrease moderately early after infection and are maintained at

an elevated level through the course of disease.33 Only at thevery late stages of AIDS do they decline. Thus, the diminishedactivity seen in most symptomatic individuals cannot be ex¬

plained by the loss of CD8+ lymphocytes. This conclusion issupported by results which indicate that the degree of antiviralactivity does not correlate with the peripheral blood CD8+lymphocyte count (unpublished observation).

Closer examination of the CD8+ cell population in HIV-infected individuals has revealed alterations in several subsets ofCD8+ cells.32 In particular, percentages of CD8+ cells thatexpress CD38, CD57, or HLA-DR increase early after infectionwith HIV and usually continue to increase over time. Con¬versely, the proportion of Leu8+ CD8+ cells decreases afterinfection. These alterations in the levels of CD8+ cell subsetsprompted the hypothesis that if CD8+ cell anti-HIV suppressiveactivity is exhibited only by a specific subset of CD8+ cells, thenprogressive loss of this subset might lead to a diminished level ofantiviral activity, and subsequent onset of symptoms.

To test this hypothesis, we evaluated whether CD8+ cellsubset changes during HIV infection correlated with changes inthe anti-HIV activity. A cross-sectional study employing dual-color flow cytometry in conjunction with functional studies was

utilized. CD8+ cell subsets examined included CD38, CDllb,HLA-DR, Leu8, CD57, and CD45RA. The relationship be¬tween the amount of these subsets, the clinical status, and thelevel of anti-HIV activity was determined.27 In agreement withpreviously reported findings,30-32 elevated levels of the CD38,CD57, and HLA-DR subsets were found in all clinical stages ofinfection tested; asymptomatic, symptomatic non-AIDS, andAIDS patients when compared to HIV seronegative controls. Incontrast, the Leu8 and the CD45RA subsets were decreased,although only slightly in the latter case.

This study also revealed that the amount of CD1 lb+ CD8+cells in AIDS patients was on the average three times higher thanthat in either of the other groups. Expression ofCD 11 b on CD8 +

cells from asymptomatic, symptomatic non-AIDS, and AIDSpatients was found to be 9%, 11%, and 32%, respectively. But,more important, this finding also indicated that the relativenumber of CDllb- CD8+ cells was decreased in AIDS pa¬tients. Nevertheless, based strictly on numerical value, thisdifference alone would not appear to be sufficient to explain thevariations in functional activity exhibited by the different clini¬cal groups.25

However, when the level of anti-HIV functional activity was

compared with the relative number of CD8+ cells expressingthese various markers, a significant correlation was foundassociated with the CD1 lb- and HLA-DR"1" subsets. Individu-


als that possessed CD8+ cells that were <90% CDllb",exhibited poor antiviral function, while all those individualswith >90% CD8+ CDllb- cells showed moderate to stronganti-HIV activity.27 As for HLA-DR coexpression, those indi¬viduals with >75% CD8+ HLA-DR+ cells exhibited moderateto strong activity, whereas those with <75% CD8+ HLA-DR"1"cells had weak to moderate activity. The lack of correlationamong subjects who possess intermediate numbers of CD8 +

HLA-DR cells suggests this activity may reside in a particularsubset of the CD8 + HLA-DR+ cells. In contrast to theseobservations, Bettens et al. were unable to find a good correla¬tion between the inability of PMC to replicate HIV (presumeddue to CD8+ cell-suppressive activity) and the percent of CD8+HLA-DR"1" cells in the patient.34 Unfortunately, the use of PMCin their assays makes it difficult to discern between the antiviralactivity of the CD8+ cells and other possible antiviral activitiesmediated by other mononuclear cells.

To directly determine which subset(s) of CD8+ cells exhibitthe antiviral activity, we examined responses from CD1 lb- andHLA-DR+ subsets of CD8+ cells as well as the other CD8+subsets shown to change upon infection with HIV. Each subsetwas obtained from purified CD8+ cells using either positive or

negative selection with immunomagnetic beads. In repeatedexperiments, similar levels of anti-HIV activity were exhibitedby CD8+ cells expressing CD45RA, CD38, and CD57, as wellas those that did not.27 However, upon examination of prepara¬tions of CD8+ cells depleted or enriched for CD1 lb, anti-HIVactivity against naturally or acutely infected CD4+ cells corre¬

lated with the CDllb" subset. In addition, while CD8+ cellsthat expressed HLA-DR+ suppressed HIV replication in eitherassay, CD8+ cells depleted of the HLA-DR4" subset exhibitedvery little antiviral activity.

Expression of CD1 lb has been correlated with a suppressoractivity of CD8+ cells, and absence of this marker associatedwith cytotoxic function by CD8+ cells.35 Hence, CD8+ cellanti-HIV activity appears to reside predominantly in CD8+ cellsthat express an activated or cytotoxic phenotype. Whether thesecells represent a single subset that expresses HLA-DR and not

CD1 lb or mutually exclusive subsets remains to be eluciated.A similar conclusion of a.cytotoxic phenotype for the antiviral

CD8+ cells was reached by Tsubota et al.16 when examiningsubsets in the SIV system. Their conclusion was based on theability of anti-LFA-1 and certain anti-CD8 antibodies to allowfor HIV replication in mitogen-stimulated PMC from HIV-infected individuals, and on their finding that the antiviralactivity resided in CD29+ CD45RA" LFA-1+ CD8+ cells,markers associated with cytotoxic cells. It is clear that more

work is needed in this area in order to define which CD8+ cellsexhibit this activity. If an active subset can be defined, an easymethod for assessing the antiviral potential in HIV-infectedindividuals could be developed using flow cytometry.

In summary, these studies indicate that, although the correla¬tion between CD1 lb absence or HLA-DR expression on CD8+cells and the clinical status or functional activity is statisticallysignificant, it is not absolute in either case. Thus, it is difficult toconclude from these observations whether the diminished anti¬viral activity in symptomatic non-AIDS and in AIDS patients isdue to the loss of the CD8+ cell subset(s) that primarily exhibitsthis activity. Possibly the absence of certain CD8+ cell subsets is

D Autologous HIV-1 Seropositive CD8+ cells Heterologous Sero negati ve CD 8+ cells

1.0

0.5 <ut-J 0.25— LUL)l··^.LU

0.1

0.05- I 1EXPERIMENT

FIG. 4. Extent of anti-HIV activity by CD8+ cells frominfected and uninfected individuals as measured by the endoge¬nous assay. In each experiment (A- ), the extent of CD8+ cellanti-HIV activity was assessed by determining the effector/target (E/T) cell ratio necessary to suppress HIV replication innaturally infected CD4+ cells by 90% or greater. Open barsrepresent results obtained when CD8+ lymphocytes from anHIV-infected individual were titrated against autologous CD4+cell targets. Dark bars represent results obtained when heterol¬ogous CD8"1" lymphocytes from an HIV seronegative individualwere titrated against the CD4+ cell targets.

only one of the contributing factors responsible for the reductionin this CD8+ cell anti-HIV activity (see below).

CD8+ cell response in uninfected individuals

Studies of uninfected individuals have indicated that afterstimulation with PHA, CD8+ cells from up to 80% of seroneg¬ative individuals can suppress HIV replication in heterologousnaturally infected CD4+ lymphocytes (the endogenous assay)(unpublished observation). Brinchmann et al. have also notedthis type of antiviral activity associated with anti-CD3-stimu-lated CD8+ cells from uninfected individuals.17 In our studies,the extent of the antiviral response exhibited by CD8+ cells fromseronegative individuals was generally lower than that of HIV-infected individuals; often twofold, but sometimes as much as

10-fold (Fig. 4). This finding could indicate that CD8+ cellsfrom HIV seronegative individuals utilize a different mechanismto inhibit virus replication, or that seronegative individuals havea lower frequency of CD8+ cells with this type of activity.Whether CD8+ cells from seronegative individuals are strictlydependent on mitogen activation to elicit this activity remains tobe determined.

When the acute assay is used, however, CD8+ cells fromuninfected individuals do not show any anti-HIV activity, even

at high effector ; target cell ratios.23·25 This observation was

made whether autologous or heterologous CD4+ target cellswere used. Furthermore, CD8+ cells from individuals witheither chronic fatigue syndrome (a disease having activatedCD8+ cells),36 acute Epstein-Barr virus infection, or followingvaccinia immunization do not show anti-HIV activity in thisassay (these authors and Walker C, unpublished observation).Similarly, CD8+ cells from SIV infected, but not uninfected,


Table 2. Antiviral Activity Mediated by CD3+ Cells from Infected andUninfected Humans and Primates

Humans Chimpanzees Rhesus monkeysHIV HIV SIV

Infected Uninfected Infected Uninfected Infected UninfectedEndogenous assay" +Acute assay0 +

+ ++

++

++

a Purified CD4+ lumphocytes from naturally infected individuals served as target cells.b Purified CD4+ lumphocytes from uninfected donors were acutely infected with HIV-1 to

serve as target cells.

rhesus monkeys suppress virus replication in an acute assay.21It is presently unclear why these two assay systems (endogenousversus acute) yield distinctly different results with CD8 + cellsfrom uninfected individuals. This finding may reflect a differentmechanism involved in the inhibition of virus replication.

With chimpanzees, however, a different observation has beenmade. Clinically healthy, uninfected animals, never exposed toHIV, appear to have a natural CD8+ cell response against thevirus.22 Up to 50% of the animals examined showed substantiallevels of this antiviral response when assayed by the acute assay.The reason for this effect is unknown, but the response may beinvolved in the resistance of chimpanzees to HIV-induceddisease. Table 2 summarizes the above described similaritiesand differences between the acute and endogenous assays usedto detect antiviral activity in infected versus uninfected humans,chimpanzees, and rhesus monkeys.

The ability of the acute assay to distinguish infected fromuninfected individuals, has been helpful in evaluating individu¬als who are seronegative, but have a history of potentialexposure to HIV. In these studies, we have noted eight infantsborn of seropositive mothers, who have CD8+ cells that sup¬press HIV replication in culture. None of them had virus asdetermined by polymerase chain reaction (PCR) and virusisolation procedures and they remain HIV negative over a yearlater (these authors and F. Hseuh, unpublished observation).Conceivably, these infants·were exposed perinatally to virus or

viral antigen and did not sustain an actual infection. Several HIVnegative adult subjects with a high risk for infection have beenevaluated as well, and only one has expressed CD8+ cellanti-HIV activity, suggesting exposure to HIV or its proteins.These studies could provide a potential method for detectingexposure to HIV in the absence of antibody or any otherevidence of virus infection.

MECHANISM OF ACTION

HLA compatibility requirementsAs mentioned above, heterologous CD8+ lymphocytes from

one HIV-infected individual can suppress HIV replication innaturally infected CD4+ cells from another HIV-infected indi¬vidual. 17·25 In addition, CD8+ cells from HIV-infected individ¬uals23·25 as well as CD8+ clonai cell lines37 inhibit HIVreplication in acutely infected heterologous CD4+ cells from

seronegative donors. These findings have been made routinelyin greater than 15 and 70 separate experiments, for naturally andacutely infected cells, respectively. Although these cell donorsin most cases were not HLA typed, the probability is high that inmany of these experiments, if not most, the cells were mis¬matched. If this conclusion is correct, it would suggest that thisCD8+ cell-mediated anti-HIV activity is not HLA restricted, atleast not at the effector phase, though possibly at the inductionphase.

There is also no evidence that the antiviral activity mediatedby CD8+ cells in HIV-1-infected chimpanzees is MHC re¬

stricted.22 However, in the SIV-infected rhesus monkey model,Tsubota et al.16 provided evidence suggesting, though notformally proving, that CD8+ lymphocytes were incapable ofinhibiting SIV replication in MHC class I mismatched PMC.The reason for this discrepancy requires further study.

Noncytotoxic mechanism

Classically, CD8+ cells are thought to function during viralinfections by exhibiting a cytotoxic mechanism that lyses in¬fected cells in an antigen-specific MHC class I restricted fash¬ion.38-41 In HIV infection, CD8+ cells that show this type ofactivity are known to be present and have now been wellcharacterized (reviewed in Ref. 11). One major differencebetween cytotoxic and "suppressing" anti-HIV CD8+ lym¬phocytes is that in the latter case, the CD8+ cells operate in theapparent absence of cell killing, and without requiring directcontact (i.e., conjugate formation) (see below).

The first line of evidence against lytic activity by the suppress¬ing CD8+ lymphocytes came from the observation that highlevels of virus replication developed after the removal of CD8 +

cells from the PMC cultures of infected asymptomatic individ¬uals in which no RT activity was previously detected.12 Thisfinding indicated that infected cells were still present andcapable of replicating HIV. Other studies showed that nearlyidentical numbers of CD4+ cells remained in CD4+ cell cultureswith or without CD8+ cells, even though virus replication was

suppressed >90% in the former cultures.18 Enumeration of theinfected CD4+ cells by IFA 13 days after the removal of CD8 +

cells revealed similar numbers of infected cells in the cellcultures with or without CD8+ cells. Thus, these studiesdemonstrated that the CD8+ cells were neither inhibiting prolif¬eration nor killing the CD4+ cells.


In a separate study,42 naturally infected CD4+ cells were

cultured until they were actively replicating HIV; then CD8 +

cells were added to suppress virus replication. Four days later,the number of infected CD4+ cells present in these cocultureswas determined by an infectious center assay involvinga limiting dilution procedure. Although HIV replication was

drastically suppressed in these cultures, 10-30% of the CD4+cells contained infectious virus, approximately the same numberfound in CD4+ cells cultured alone.

Similar observations were made when acutely infected CD4+cells were used as targets,24 with the exception that often thenumber of infected cells slightly increased in the presence ofCD8+ cells. In these acute assays, virus spread to uninfectedcells could be demonstrated even though RT activity was

reduced to very low levels. Whether this result could beattributed to low-level production of virus in the culturesescaping suppression by CD8+ cells or to an inability of theCD8+ cells to prevent HIV spread is unclear. Nevertheless, theabove studies indicate that CD8+ cells can suppress HIVreplication in acutely as well as naturally infected CD4+ lym¬phocytes without eliminating infected cells.

A further piece of evidence against a lytic mechanism came

from 51Cr release assays examining the effect of purifiedPHA-stimulated CD8+ cells on 51Cr-labeled seronegativeCD4+ cells acutely infected with HIV-1.24 The antiviral activityof the CD8+ cells did not correlate with the ability to lyseHIV-infected or uninfected targets. Along these lines, Koeniget al.43·44 have assessed the ability of HIV-specific, HLA-restricted cytotoxic CD8+ cell clones to inhibit HIV replicationin culture. Only a small portion of these clones was able to

strongly suppress replication. This activity was dependent on themethod of stimulation; antigen but not mitogen. An even smallerportion of these clones was able to suppress HIV replicationthrough a semipermeable membrane (i.e., soluble factor medi¬ated) and only to a limited extent.

Role of a soluble factorWhen investigating the requirements for cell to cell contact,

Walker and Levy found that the CD8+ cells can suppress HIVreplication in purified naturally infected CD4+ cells, even ifseparated by a semipermeable membrane in transwell devices.45This finding provided the first evidence that cell to cell contactbetween the CD4+ and the CD8+ cells is not necessary to inhibitHIV replication. We have subsequently shown that HIV repli¬cation in naturally infected CD4+ cells can be suppressed by theaddition of filtered supernatants from CD8+ cell cultures (un¬published observation). Work by Brinchmann and colleaguesconfirmed these observations and went on to show that theCD8+ cell culture fluids did not affect CD4+ cell prolifera¬tion.17 It has been difficult to demonstrate anti-HIV suppressiveactivity using transwells in the acute assay,24 but recently we

observed that filtered medium from CD8+ cell cultures can

suppress HIV replication by 50-85% in a microtiter acute assay(unpublished observation).

Whether the activity of this factor is solely responsible for theanti-HIV effects of CD8+ cells is not known since CD8+ cellsare much more efficient at inhibiting HIV replication when indirect contact with the CD4+ cells.45 As much as 10-fold moreCD8+ cells are needed when using the transwell device to obtain

Table 3. Effect of Various Cytokines on HIVReplication in CD4+ Lymphocytes3

Cytokine Endogenous assay' Acute assay

c

IL-1IL-2IL-3IL-4IL-5IL-6IL-7IL-8IL-9IL-10G-CSFGM-CSFTNFaIFNaIFNßIFN7TGFß

Slightly inhibitsEnhancesNo effectDelaysd-eEnhancesNo effect6EnhancesInhibitsNo effectNo effectNo effectEnhancesInhibitseInhibitseNo effect6No effectInhibits

No effectEnhancesNo effectDelaysdeSlightly enhancesNo effect"No effectNo effectNot testedNo effectSlightly enhancesNo effectEnhances/inhibitsfInhibits6Inhibits6No effectInhibits6

"Each cytokine was tested over a range of concentrations,from saturation (usually 100 U/ml) to a 1 ; 100 dilution. Thestated effect of the cytokine was dose dependent in each case.

b Cytokine was added to naturally infected CD4+ cells at theinitiation of the culture and replenished daily on every third day.

6 Immediately after the CD4+ cells were acutely infected withHIV-1SF33, the cells were cultured with cytokine. The cytokinewas replenished daily or every third day.

d IL-4 delays the development of peak HIV replication by 3 to6 days compared with untreated CD4+ cells.

6 Antibodies to this cytokine do not block CD8+ cell anti-HIVactivity.

f Enhances at high concentrations and inhibits at subsaturatingconcentrations.

an equivalent degree of suppression (unpublished observation).Furthermore, CD8+ cells from some HIV-infected individualsdo not suppress HIV when separated from the CD4+ cells in thetranswell device, even at high effector/target cell ratios. Yet,they inhibit replication when placed in direct contact with theCD4+ cells.45

A major effort has been directed to the identification andcharacterization of this anti-HIV 'lymphokine' produced byCD8+ cells. Our initial approach examined a large panel ofrecombinant human cytokines for identity to the CD8+ cellanti-HIV factor.46 Each cytokine was tested for its ability toinhibit HIV replication in naturally or acutely infected CD4+cells. In addition, monoclonal antibodies directed against vari¬ous cytokines were employed in attempts to neutralize theanti-HIV activity in CD4+/CD8+ cell cultures.

Of the 15 different cytokines evaluated, only TGFß, TNFa,and IL-8 were found to consistantly suppress HIV replication inCD4+ cells in a dose-dependent manner (Table 3). CD8 + cellsproduce TNFa47 and TFGß47a (Jan Devries, personal commu¬

nication), but it is not known if they can produce IL-8. However,antibodies directed to TNFa and TFGß, as well as IFNa and ßand TNFß did not block anti-HIV activity from CD8+ cells.Moreover, no 2'-5'A synthetase activity was detectable in thetarget CD4+ cells exposed to CD8+ cells,46 further suggestingthat an IFN is not responsible, despite their known inhibitory


activity against HIV replication.48'49 Recently, Brinchmann et

al. reported that TNFa and IFNa, ß, and y, are not responsiblefor this CD8+ cell activity based on the inability of monoclonalantibodies specific for these cytokines to abrogate the antiviralactivity of CD8+ cells.50 This activity, therefore, appears to bemediated by a novel cytokine. The biophysical nature of thiscytokine and whether it is exclusively made by CD8 + cells are

currently under investigation.

Role of adhesion molecules in cell to cellcontact suppression

As noted above, cell to cell contact is not necessary for theinhibition of HIV replication in CD4+ cells; however, the mostefficient block of HIV replication occurs when the effector andtarget cells are placed in direct contact. In studies examiningdirect cell to cell interaction between CD4+ and CD8+ cells,antibodies to the CD3 subunit of the T-cell receptor (TCR) and toa number of cell surface molecules involved in cellular adhesionwere examined for their ability to abrogate the anti-HIV suppres-sive activity of CD8+ cells. Antibodies to CDlla (LFA-1),CD18, CD58 (LFA-3), CD44 (Hermes), Dreg 2 (homing recep¬tor), CD8 (Leu2a), or CD3 (Leu4) were unable to substantiallyand consistently block the antiviral activity of CD8+ cells(unpublished observation). This observation was made regard¬less of whether naturally or acutely infected cells were used as

targets with the exception that some inhibition of CD8+ cellactivity was noted with antibodies to CD8 and CD3 in the acute

assay.24 Brinchmann et al. have noted that monoclonal antibod¬ies specific for LFA-1 (7G5G1) or CD8 (ITI-5C2) do not blockCD8+ cell anti-HIV activity in an assay similar to the endoge¬nous assay.17 Thus their findings are in agreement with ours.

In contrast to these results, Tsubota and colleagues reportedthat addition of antibodies specific for LFA-1 (2F12), as well as

certain anti-CD8 antibodies (not Leu2a), to naturally infectedPMC cultures allowed for HIV-1 replication. '6 They concludedthat these antibodies were blocking the ability of the CD8+ cellspresent in PMC cultures to inhibit virus replication in theinfected cells. It is likely these different results reflect thevarious antibodies and concentrations used. Further studies are

needed to clarify the discrepancies in these studies regarding thenecessity for interaction between these surface molecules on theeffector cells and their ligands on the target cells.

CD8+ cells block HIV RNA expressionStudies discussed above indicate that the antiviral response of

CD8 + cells does not involve the elimination of infected cells nor

does it inhibit the activation of CD4+ cells as evidenced bynormal levels of proliferation and activation marker expression.In attempts to determine how the CD8+ cells (or their solublefactor) inhibit HIV replication we investigated which step in theHIV replicative cycle was affected. Naturally infected CD4+cells were cultured alone until they were actively replicatingHIV. Then CD8+ cells were added to suppress virus replication.Every two days thereafter, HIV protein and RNA expressionwere measured by IFA and in situ hybridization, respectively.On the sixth day, the number of infected CD4+ cells present inthe cocultures was determined by an infectious center assay.42Both the expression of HIV protein and RNA was markedly

inhibited by CD8+ cells; often >90% reduction occurred com¬

pared with expression in CD4+ cells cultured alone. Impor¬tantly, infectious center assays indicated that the number ofinfected CD4+ cells in the cultures with CD8+ cells was similarto that in cultures without CD8+ cells. These results confirmedthat the CD8+ cells were not eliminating infected cells andindicated that HIV messenger RNA is reduced. They did notdiscern, however, whether transcription is blocked or transcriptsare being rapidly degraded.

In the SIV-rhesus model, Kannagi and co-workers were ableto demonstrate that CD8+ cells block the accumulation ofunintegrated DNA26 and in an earlier study, found that CD8 +

cells do not impair the ability of HIV to bind to CD4+ cells.21 Ifthese two systems are comparable, the findings suggest that theblock is postbinding yet pretranslational. We are currentlytesting the ability of CD8 """cells (or their culture supernatants) toinhibit íü/-mediated transcription in LTR-CAT transfectedPMC. These experiments should determine if HIV transcriptionvia the LTR can be inhibited by CD8+ cells.

SPECIFICITY OF CD8+ CELL-MEDIATEDANTIVIRAL RESPONSE

CD8+ cytotoxic cells from HIV-infected individuals havebeen shown to be specific for multiple epitopes within most ofthe HIV proteins, structural and regulatory. These include thegag, env, nef vif, and pol gene products (reviewed in Refs. 11and 51). The specificity of these anti-HIV CTL responses isthought to be regulated by HLA antigens.52 In contrast, to date,no antigen specificity has been demonstrated for CD8+ cells thatmediate the inhibition of HIV replication in vitro. CD8+ cellsfrom one individual can suppress HIV replication in heterolo¬gous CD4+ cells from any of several other individuals. More¬over CD8+ cells from a large number of different individualscan inhibit replication in CD4+ cells acutely infected with one

HIV-1 strain (SF33) (see above). These observations suggestthat if specificity to HIV exists, then it must be to a highlyconserved epitope(s). This conclusion could also explain howCD8+ cells from an HIV-1-infected individual can suppressviral replication in CD4+ cells acutely infected with HIV-1 or

HIV-2, or even SIVmac.23 In addition, CD8+ cells from chim¬panzees infected with HIV-1SF, can inhibit replication offflV-l--,.0

CONCLUSIONS

We have summarized the efforts of our laboratory and othersto characterize the anti-HIV-suppressing activity exhibited byCD8+ lymphocytes. The only other well-characterized antiviralactivity demonstrated with CD8+ cells from HIV-infected indi¬viduals is an antigen-specific, HLA-restricted cytotoxic re¬

sponse. At present, whether these two activities are mutuallyexclusive is unclear, but most of the evidence suggests that theyare separate.

Table 4 lists the important characteristics of the suppressingtype of antiviral response. This activity is a property of HLA-DR+ and/or CD1 lb-, CD8+ cells (not macrophages or anyother type of lymphocyte) and is dose dependent. It does not


Table 4. Properties of CD8+ Cell ViralSuppressing Activity

Active against HIV in naturally or acutely infected CD4+cells

Observed with CD8+ cells from infected humans andnonhuman primates

Can be detected in uninfected individuals when usingnaturally infected CD4+ cells

Dose dependentNonlytic mechanismInvolves a soluble factorOptimal with celkcell contactMHC unrestricted at effector levelDoes not block activation or proliferation of CD4+ cellsBroad specificity (antigen nonspecific?)Exhibited predominantly byHLA-DR+ and CDllb- CD8H

cell subsetsBlocks HIV RNA expressionCorrelates with healthy clinical state and high CD4+ cell

count

require HLA compatibility and operates via a nonlytic mecha¬nism that blocks the expression of HIV RNA in infected CD4+cells. The effector phase is broadly reactive: CD8+ cells fromone individual can suppress replication of a variety ofHIV-1 andHIV-2 strains, and therefore it is uncertain whether any antigenspecificity exists. This phenomenon may be a consequence ofthe CD8+ cell antiviral cytokine acting in an antigen-nonspe¬cific manner. Both cross-sectional (point prevalence) and longi¬tudinal studies indicate that this anti-HIV activity has clinicalrelevance.

Human subjects (unpublished observation) and rhesus mon¬

keys21 develop activity soon after infection but, in the case ofhumans, lose the activity over time, often subsequent to progres¬sion to disease. Thus, CD8-mediated anti-HIV activity can beadded to the growing list of defects related to CD8+ lympho¬cytes described in AIDS, including; decreased antigen53 andmitogen responsiveness,34·54 decreased HIV-specific CTL fre¬quency,55·56 decreased celj-mediated responses against a vari¬ety of virsuses,57·58 and loss of spontaneous suppressor cellactivity.59

The accumulated evidence strongly suggests that this naturalresponse mediated by CD8+ lymphocytes is important in pro¬tecting the host against the deterioration of the immune systemby HIV. One important consideration is whether antiviraltreatments would be harmful to this activity. Our preliminarystudies on the effect of AZT indicate that HIV-infected individ¬uals on this drug for more than one year show no consistentchange in their CD8+ cell antiviral response (unpublishedobservation).

Why this CD8+ cell antiviral activity diminishes with time isnot known. Several explanations can be offered. (1) The CD8+cells may be losing function due to a lack of CD4+ cell help; a

consequence of the loss of CD4+ cells over time. As discussedabove, the extent of CD8+ cell-mediated anti-HIV activitydirectly correlated with CD4+ cell numbers in the infectedindividual. (2) An accumulation of toxic effects of HIV productsin the body may cause reduced CD8+ cell activity. (3) EventualHIV infection of CD8+ cells60'61 could disturb cellular func¬

tion, although we cannot detect HIV in CD8+ cells by PCRanalysis (these authors and L. Pan, unpublished observation).(4) Certain cells in HIV-infected individuals have been shown tobe capable of suppressing CTL activity.62 Perhaps similar cellsare capable of suppressing the CD8+ cell anti-HIV activity we

are describing. (5) Chronic activation32 may lead to the devel¬opment of an unresponsive state in the CD8+ lymphocytes.

All of the above explanations could result in the loss of a

CD8+ cell subset(s) or decreased antiviral factor productionfrom the CD8+ cells, thus resulting in the diminished activityobserved in AIDS patients. The answer to this important ques¬tion could help in the development of therapeutic approaches tomaintain this natural antiviral response in infected individuals.

ACKNOWLEDGMENTS

The research studies by the authors cited on this review were

supported by NIH Grants ROÍ AI30350 and AI24286, HooperFoundation Grant CA09043-17, and a grant from the Universi¬tywide State AIDS Research Program.

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Address reprint requests to:Carl Mackewicz

Department of MedicineCancer Research Institute

University of California School of MedicineSan Francisco, CA 94143-0128

Documents

CD8 + Cell Anti-HIV Activity: Nonlytic Suppression of Virus Replication