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Viruses and immune systems play microscopic gamesof ‘hide-and-seek’ during the course of an infection.The virus attempts to find and enter its host cell, repli-cate its genome, assemble new particles and spread tonew target cells, while minimizing its exposure to theimmune system. At the same time, the immune systemattempts to recognize and eliminate the invadingviruses as quickly as possible and without causingdamage to the host. In cases of chronic viral infections,or when viruses (such as retroviruses) use cells of theimmune system as their hosts, this game can be verycomplicated indeed.

To gain an advantage, the immune system usesoverlapping effector mechanisms, which are activatedby viral infection and suppress one or more steps in thevirus life cycle (FIG. 1). NATURAL KILLER CELLS (NK cells)1,2,granulocytes and, possibly, γδ -T CELLS3–5 provide the ini-tial line of defence when stimulated by chemical signalsthat are released at sites of infection. All of these cellsproduce immunoregulatory molecules: neutrophilsproduce antimicrobial defensins; and NK cells and γδ-T cells can kill infected cells through cytolyticmechanisms1–5. Host macrophages and dendritic cellsalso have important roles in this early stage by takingup and presenting viral antigens and by secreting several soluble CYTOKINES and CHEMOKINES that help toamplify the immune response. These early processes arecalled INNATE IMMUNE RESPONSES because they can act

immediately using extant receptors and do not requirethe induction of major histocompatibility complex(MHC) gene products or antigen presentation in thecontext of MHC molecules. Over time, the innateresponse gives way to an adaptive response, whichrequires viral antigen presentation by cell-surfaceMHC molecules. Adaptive immunity is carried out byseveral effector-cell subsets, including CD8+ T CELLS, CD4+

HELPER T CELLS, and B cells. These cells establish importantcell–cell communication mechanisms by releasing awide range of soluble molecules. The cellular arm ofadaptive immunity is active at an early stage (~1 weekpost-infection) and has an important role in fightingmost viral infections. CELLULAR IMMUNE RESPONSES aremediated by CD8+ cytotoxic T lymphocytes (CTLs)that have been primed by dendritic cells and other cellsubsets that present viral antigens in conjunction withClass I MHC molecules. Once primed by MHC–antigencomplexes and co-stimulatory signals, CTL clones targetinfected tissues and attempt to kill infected cells beforethey can produce progeny viruses. To do this, the CTLmust interact with viral antigens in conjunction withClass I MHC molecules on the surface of an infectedcell. The CTL then delivers pro-apoptotic signals andsoluble cytolytic enzymes that destroy the infectedtarget. Several weeks after infection, the humoral armof adaptive immunity joins the cellular response incombating infection. HUMORAL IMMUNE RESPONSES arise as a

CONTROL OF HIV-1 INFECTION BY SOLUBLE FACTORS OF THEIMMUNE RESPONSEAnthony L. DeVico and Robert C. Gallo

An increasing body of evidence indicates that the immune system uses a range of solublemolecules to suppress certain viral infections without killing infected host cells. Recent studiesindicate that such factors might have an especially important role in the immune response to HIV-1. Accordingly, this review uses HIV-1 as a model to explore the diversity of non-cytolyticantiviral factors and considers how these molecules might be used to develop new therapeuticand prophylactic strategies to fight viral infections.

NATURAL KILLER CELLS

(NK cells). Lymphocytes that donot express the T-cell receptor(TCR) or B-cell receptor (BCR)and which mediate natural killing against prototypical NK-cell-sensitive targets.

γδ-T CELLS

T lymphocytes express a T-cell receptor (TCR) that iscomposed of either α- andβ-subunits (αβTCR) or a TCRthat is composed of γ- and δ-subunits (γδTCR). Most(>90%) T cells have an αβTCRthat recognizes conventionalMHC class I or II ligands. T cellsexpressing γδTCR are lesscommon and the ligands of thistype of receptor are less wellcharacterized.

Institute of Human Virology,University of MarylandBiotechnology Institute,Baltimore, Maryland 21202,USA.Correspondence to R.C.G.e-mail: gallo@umbi.umd.edudoi:10.1038/nrmicro878

CYTOKINES

Originally used to describe agroup of immunomodulatorygrowth factors, the termcytokine is now used to describea diverse group of solubleproteins that modulate the manyactivities of cells and tissues.

CHEMOKINES

Chemotactic cytokines involvedin specific inflammatoryresponses. They aredifferentiated into CC or CXCchemokines on the basis of theirprimary amino acid sequence.

INNATE IMMUNE RESPONSE

The first line of defence againstmicrobial infections. Innateresponses do not require theinduction of MHC geneproducts or antigen presentationin the context of MHCmolecules.

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infection, but also activate a number of intracellularpathways that directly suppress viral replication withoutkilling the host cell6–18. Interferons have been shown tosuppress hepatitis B virus (HBV), hepatitis C virus(HCV), herpes simplex virus, vesicular stomatitis virus(VSV), vaccinia virus, picornaviruses, retroviruses,influenza viruses and other types of viruses in vitro in anon-cytolytic manner6–13,17. These broad antiviral effectsare mediated by several mechanisms that rely on recep-tor-mediated gene-expression pathways, including theJAK/STAT (Janus kinase/signal transducer and activa-tor of transcription) signal-transduction cascade6–11,18,19.In the presence of double-stranded RNA, IFN-α andIFN-β mediate well-characterized antiviral mecha-nisms that either degrade viral RNA transcripts orinhibit viral protein synthesis. Other mechanismsinhibit the attachment, entry or assembly of certainviruses. IFN-γ is likely to activate overlapping pathwaysas well as non-redundant pathways and comparableantiviral mechanisms8,11,18,19. Second, many cell subsetsof both the innate and adaptive responses, includingNK cells, mononuclear phagocytes, γδ-T cells, CD4+ Tcells and CTLs, have been shown to secrete non-cytolytic

result of interactions between antigen-specific B cellsand CD4+ helper T cells that have been stimulated byviral antigens in conjunction with MHC Class II antigens.Ultimately, the interacting B cells release antibodies thatreact with specific epitopes on the viral antigens. In mostcases, antiviral antibodies prevent the intercellulartransmission of virions and the reinfection of the host,although in some instances they can also contribute tothe direct suppression of viral replication. Overall, theconventional view of the immune system holds thatthe control and prevention of viral infections restswith one or more of these defence mechanisms,depending on the virus in question.

However, this view is being reconsidered. We arebeginning to understand that both innate and adaptiveresponses to viral infections can be supplemented byNON-CYTOLYTIC SOLUBLE SUPPRESSOR FACTORS, which, remark-ably, can be antiviral either by accident or design. Thisnew perspective has emerged primarily from four linesof evidence. First, it has become clear that certainimmunoregulatory cytokines, including INTERFERONS

(IFNs) and tumour necrosis factor (TNF), not onlyinduce apoptosis and necrosis of certain cell types on

Innate response

γδ -T cells NK cells

Host cellVirus

Granulocytes

Adaptive responseAntigen-presenting cells (dendritic cells, macrophages) Antigen-presenting cells (dendritic cells, macrophages)

CD8+ T cells

Cellular Humoral

CD4+ T cells B cell

DefensinSoluble non-cytolytic factors(IFN, TNF-α, certain CC

chemokines in the case of HIV-1, other molecules)

Cytolytic cell killing Cytolytic cell killing AntibodiesSoluble non-cytolytic factors(IFN, TNFα, certain CC

chemokines in the case of HIV-1, other molecules)

Figure 1 | A working model of an antiviral immune response that uses cytolytic cell killing, antibodies and soluble non-cytolytic factors as a means tosuppress infection. In the first phase of an infection, viruses infect susceptible host cells. In response to infection, the innate immune response mediates severalantiviral mechanisms, including cytolytic cell killing by NK cells, which lyse infected cells. Several soluble factors are also released, which directly suppress infectionwithout killing infected host cells. Infected cells or virions that escape the innate response are controlled by the humoral and cellular adaptive immune responses.However, non-cytolytic antiviral mechanisms continue to have a crucial role in suppressing viral replication. Infected host cells are shown in red and uninfected cells ingreen. IFN, interferon; NK, natural killer; TNF, tumour necrosis factor.

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co-challenged with LCMV and the related pichindearenavirus. Although the animals were protected fromLCMV, they were still infected by pichinde39. Similarly,passive transfer of CTLs that are specific for influenzahaemagglutinin HA

2protected mice from influenza

virus but not from concomitant challenge withinfluenza haemagglutinin HA

1(REF. 40). Such specificity

indicates that if soluble non-cytolytic factors are releasedby antiviral CD8+ T lymphocytes, they might be activeonly over very short distances. On the other hand, itwas shown that hepatocellular HBV gene expressionwas potently suppressed during LCMV infection inHBV transgenic mice41. Such suppression was non-cytolytic and principally mediated by TNF-α andIFN-α/β produced by LCMV-infected intrahepaticmacrophages41. Similarly, clinical studies have pro-vided evidence that HBV replication is suppressed byacute hepatitis A virus (HAV)-induced production ofsoluble factors including IFN-γ 42,43. So, soluble‘bystander’ suppression may indeed occur morereadily in certain tissues and viral systems.

In recent years, HIV-1 has been characterized as avirus that is highly sensitive to non-cytolytic suppres-sion by soluble factors. Indeed, the nature of solubleHIV-1 suppressor activity might reflect an intimatelink between viral replication and the immune system.So, HIV-1 infection is an ideal model for appreciatingthe capacities of soluble factors to mediate antiviralimmunity. Accordingly, the following sections focuson HIV-1 suppressor factors, their activities and theirrelevance to natural infection.

Soluble HIV-1 suppressor activityThe first observations of non-cytolytic HIV-1 suppressoractivity were made almost two decades ago in thecontext of CD8+ T-cell responses44–46. At that time, itwas recognized that peripheral blood mononuclearcells (PBMCs) taken from seropositive asymptomaticindividuals often failed to manifest HIV-1 replicationin vitro. In 1986, Walker et al.44 showed that this sup-pression of viral replication was linked to the presenceof CD8+ T cells in the cultures. Selective removal ofthese cells resulted in an elevation of viral replication,whereas depletion of other cell types, such as CD16+

cells (including NK cells), had no effect44. Furthermore,reconstitution of depleted cultures with autologousCD8+ T cells re-established suppression of HIV-1replication in a concentration-dependent mannerwithout altering the proliferation or viability of CD4+

HIV-1 host cells45–49. Taken together, these data showthat CD8+ T cells are able to block active HIV-1 repli-cation through non-cytolytic virus-suppressivemechanisms. Later studies revealed two more signifi-cant characteristics of this activity. First, the factor(s)that are responsible for HIV-1 suppressor activity aresoluble45,46,50. Experiments carried out in transwellchambers clearly showed that non-cytolytic suppres-sion was achieved even when the CD8+ T cells wereseparated from the CD4+ host cells by semi-permeablemembranes50,51. Other experiments showed that HIV-1suppression was mediated by filtered supernatants from

antiviral molecules, such as IFN and TNF-α, in responseto stimulation by viral antigens10. Third, a substantialamount of data from experiments with HBV transgenicmice indicate that the primary mechanism for the controland clearance of HBV infection is provided by the directnon-cytolytic antiviral activity of soluble factors thatare released by CTLs and/or NK cells10,14,15. In thistransgenic system, the animals exhibit hepatic HBVgene expression, but do not mount an immune response(anti-self) against HBV antigens. Furthermore, the datado not support cell–cell virus spread and reinfection byHBV. So, these animals are highly useful for specificanalyses of effector subsets. Adoptive transfer experi-ments carried out in these mice showed that HBV-specificCTL clones suppress viral replication in many infectedcells through non-cytolytic antiviral activities that aremediated by IFN-γ and, perhaps, TNF-α20–23. Fourth,cell subsets other than CTLs have been shown to releasesoluble factors with non-cytolytic antiviral activity.CD4+ T cells have been shown to suppress influenza,VSV, HBV and vaccinia infections in mice24–30, andHIV-1 infections in human lymphocyte cultures31,through the release of soluble factors. NK cells havealso been observed to suppress certain viral infectionsby releasing non-cytolytic antiviral factors32–37. Theproduction of soluble suppressor factors by γδ-T cellshas been correlated with the protection of macaquesfrom simian immunodeficiency virus (SIV)38.

Given these findings, a new model for antiviralimmunity has emerged, which includes the directantiviral effects of soluble non-cytolytic factors as aneffector mechanism. In this model, the array of solublemediator factors that are released in response to infec-tion includes factors that diffuse through the site ofinfection and directly suppress virus replication with-out killing infected cells (FIG. 1). As will be discussedbelow, this working model accounts for the possibilitythat some of the virus suppressor molecules might alsoperform immunoregulatory roles. In these cases, therelative importance of antiviral versus immunoregu-latory function varies depending on the infectingvirus and the nature of the adaptive response.

In theory, this model presents three advantages forthe infected host. First, a soluble/diffusible non-cytolyticeffector function would boost the antiviral potency of aCTL beyond its destructive capacity, which is inherentlylimited by the frequency of effector–target-cell contacts.So, a CTL would be able to more rapidly clear an infec-tion in tissues where it is outnumbered by infected cells.Second, NK cells and CTLs could suppress infection invital organs without having to destroy a large number ofimportant cells. Third, an immune response to one virusmight suppress other viruses in the area by bystandereffects that are mediated through the diffusion of solubleantiviral factors. For example, the release of IFN byCTLs in response to one type of virus might partially‘sterilize’ the area to other sensitive viruses. In the pastsuch a phenomenon was difficult to show in experi-mental viral systems. In mice, CTL clones that are spe-cific for lymphocytic choriomeningitis virus (LCMV)were passively transferred to animals that were then

CD8+ T CELLS

One of the two main classes of Tlymphocytes (the other beingCD4 + T cells). CD8+ T cells exertcytolytic activity in an antigen-specific manner. They alsoproduce various cytokines toregulate the immune system.

CD4+ HELPER T CELLS

T helper cells that collaboratewith antigen-presenting cells inthe initiation of an immuneresponse.

CELLULAR IMMUNE RESPONSE

An adaptive immune responsethat is mediated by CD8+

cytotoxic T lymphocytes thathave been primed by dendriticcells and other cell subsets thatpresent viral antigens inconjunction with Class I MHCmolecules. Cellular responseshelp clear viral infections bykilling infected cells.

HUMORAL IMMUNE RESPONSE

An adaptive immune responsethat is mediated by antibodiesdirected against specific epitopeson viral antigens. Theseantibodies prevent theintercellular transmission ofvirions and the reinfection ofthe host.

NON-CYTOLYTIC SOLUBLE

SUPPRESSOR FACTORS

Diffusible molecules that arereleased during an innate and/oradaptive immune response andwhich suppress viral replicationwithout killing host cells.

INTERFERONS

(IFNs). Interferons are proteinswith potent antiviral activitythat are of particularimportance during the earlyresponse to pathogens. Type IIFNs (α, β and ω) arehomologous proteins thatinteract with a common two-chain receptor (IFNAR1and IFNAR2). Type II orimmune IFN is represented by a single protein (IFN-γ) that interacts with different two-chain receptors (IFN-γR1and IFN-γR2).

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that RANTES, MIP-1α and MIP-1β were responsiblefor nearly all of the HIV-1

Balsuppressor activity in the

CD8+ T-cell-culture supernatants. However, moreextensive testing with a wider variety of isolatesrevealed that although RANTES, MIP-1α and MIP-1βalways suppressed what were then called MACROPHAGE-

TROPIC viruses, they did not suppress T-TROPIC isolates,such as HIV-1

IIIB53. This was in contrast to unfraction-

ated CD8+ T-cell supernatants, which suppressed allstrains of HIV-1 regardless of tropism. We now knowthat macrophage-tropic viruses are selectively sup-pressed by RANTES, MIP-1α and MIP-1β owing totheir specific requirements for entry. To enter targetcells, HIV-1 must first establish an envelope–receptorcomplex that includes the viral envelope glycoprotein(gp120) and cell-surface CD4 molecule. The gp120–CD4complex that is formed by macrophage-tropic virusesthen binds selectively to a seven-transmembrane-spanning, G-protein-coupled surface co-receptor calledCCR5 (REFS 54–58), which is also the natural receptor forRANTES, MIP-1α and MIP-1β.As a consequence of thisshared receptor usage, the entry of macrophage-tropic(now called R5) HIV-1 strains is blocked by the CCR5ligands53,59–61. Conversely, the T-tropic (now designatedX4) strains enter cells using a different chemokinereceptor known as CXCR4, which is not bound byCCR5 ligands58,62 but instead by the chemokine stromal-cell-derived factor or SDF-1 (REF. 63). This preferencerenders X4 strains immune to inhibition by CCR5 lig-ands. So-called DUAL-TROPIC (R5X4) strains can use eitherco-receptor type, depending on target-cell expressionpatterns, but are inhibited by CCR5 ligands wheneverCCR5 is the operative co-receptor. So, RANTES, MIP-1αand MIP-1β collectively account for the CD8-derivedsuppression of R5 viruses (and R5X4 viruses in a CCR5-dependent system), whereas other, unknown factors areresponsible for inhibiting the isolates that must use theCXCR4 co-receptor (FIG. 3). In riposte, ‘CAF’ is nowdefined as the portion of CD8-derived suppressor activ-ity that suppresses X4 HIV-1 strains (or R5X4 strains in aCCR5-minus system), or any suppressive molecule otherthan RANTES, MIP-1α or MIP-1β17,64,65. In general, it isnow common to categorize factors according to whetherthey suppress R5 versus non-R5 (X4 or R5X4) isolates.

Second, it is clear that bulk CD8+ T cells are not theonly human cell sources of soluble non-cytolytic HIV-suppressor activities/factors (FIG. 4). For example, naiveCD4+ T cells stimulated with immobilized anti-CD3and anti-CD28 antibodies66–68 or co-cultured withantigen-pulsed dendritic cells31, NK cells stimulatedwith IL-15 and IL-12 or cytokine and anti-CD16 (REFS

35–37), γδ-T cells69, and antigen-specific CD4+ T cellclones70 have all been reported to secrete antiviralconcentrations of RANTES, MIP-1α and MIP-1βtogether with unassigned activities that suppress R5and/or X4 isolates. CD4- or CD8-depleted PBMCs thatare stimulated by live, inactivated influenza virus71

produce HIV-suppressive levels of IFN-α together withunidentified factors that suppress both R5 and X4 HIV-1isolates. Alloantigen-stimulated whole PBMC72 release apartially unassigned factor with antiviral activity that

cultures of CD8+ T cells that had been activated withmitogen or anti-CD3 antibody and interleukin (IL)-2(REFS 45,51,52). Second, the soluble factor(s) are capable ofsuppressing many, if not all, primary HIV-1 strains46,47.CD8+ cell supernatants suppressed infection in infec-tivity systems that used cell-free virus stocks, as well asin experiments that used CD4+ T cells derived fromHIV-positive individuals as the source of primary virus.

Taken together, these findings indicate that theimmunological control of HIV-1 might involve non-cytolytic antiviral mechanisms, such as those shownin FIG. 1. As CD8+ T cells release the factor(s) with sup-pressive activity, it was reasonable to suspect that thisactivity is most relevant to cellular responses againstHIV-1. So, the soluble suppressive factor was eventuallynamed CD8 ANTIVIRAL FACTOR, or ‘CAF’, on the basis of thenarrow assumption that activity could be explained by asingle molecule.

However, our perception of soluble HIV-1 suppressoractivity has progressed beyond this simplistic concept inthree significant ways. First, it is now appreciated thatsoluble HIV-1 suppressor activity reflects the collectiveaction of multiple factors. This characteristic wasrevealed in 1995, when it was determined that thechemokines RANTES and macrophage inflammatory pro-teins 1α and 1β (MIP-1α AND MIP-1β) are involved in the sup-pressor activity when released by activated CD8+

T cells53. Specifically, it was shown that neutralizinganti-chemokine antibodies completely abrogate theHIV-1 suppressor activity of activated CD8+ T cellsfrom HIV-1 seropositive, asymptomatic individuals.Notably, antibodies against any one of the chemokineshad little effect on suppressor activity against the testisolate, HIV-1

Bal. However, a mixture of antibodies

against all three chemokines reversed the suppression ofthe virus (FIG. 2). This important observation showed

CD8 ANTIVIRAL FACTOR

(CAF). The first reported solubleHIV-1 suppressor activity; CAFis released by primary CD8+ Tcells upon activation in vitro.

RANTES

A CC-chemokine that binds toand activates the chemokinereceptor CCR5.

MIP-1α AND MIP-1βLike RANTES, MIP-1α and MIP-1β are CC-chemokines thatbind to and activate thechemokine receptor CCR5. Allthese chemokines block theentry of HIV-1 strains that useCCR5 as a co-receptor.

MACROPHAGE-TROPIC

Also known as R5. A viralphenotype that is defined by theuse of CCR5 as the co-receptorfor viral entry. These isolates areselectively sensitive tosuppression by the solublesuppressor factors RANTES,MIP-1α and MIP-1β, which arenatural ligands for CCR5.

T-TROPIC

Also known as X4. A viralphenotype that is defined by theuse of CXCR4 as a co-receptorfor viral entry. These isolates arenot inhibited by RANTES,MIP-1α and MIP-1β, but aresensitive to suppression by othersoluble suppressor factors.

100

80

60

40

20

0Patient 2Patient 1 Patient 3 Patient 4

HIV

-1 e

xpre

ssio

n (%

of c

ontr

ol)

Patient HIV-SF +Anti-RANTES

+Anti-MIP-1β

+Anti-RANTES+Anti-MIP-1α+Ant-MIP-1β+Anti-MIP-1α

Figure 2 | RANTES, MIP-1αα and MIP-1ββ are primarily responsible for the suppression ofmacrophage-tropic (R5) HIV-1 replication by CD8+ T-cell-culture supernatants. In thisexperiment, the treatment of CD8 supernatants from four HIV-positive donors with a mixture ofneutralizing antibodies to RANTES, MIP-1α and MIP-1β extensively abrogates the suppression ofHIV-1BaL replication. HIV-SF, HIV suppressive factors. Reproduced with permission from REF. 53

© (1995) American Association for the Advancement of Science.

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explained by the differentiation of cells into distinctsubsets over time. As a result, the culture supernatantsreflect the presence of a dynamic collection of HIV-1suppressive molecules, some of which have yet to beidentified.

Overall, these findings indicate that both innate andadaptive immune responses use soluble non-cytolyticantiviral activity to control HIV-1 replication. Findingsthat multiple cell subsets produce factors with antiviralactivity indicate that there is a degree of redundancy insuch responses. However, it seems almost certain thatsoluble suppressor activity is always due to the actionsof multiple components (possibly providing anotherlevel of redundancy), although the nature of the com-position might vary according to the cell subset andresponse pathway in question.

The quest for other HIV-1 suppressor factorsTo fully understand the immunological significanceand practical value of soluble antiviral activity, theresponsible factors must be identified beyond RANTES,MIP-1α and MIP-1β. Accordingly, efforts have beenmade to assign a molecular identity to ‘CAF’ and HIV-1suppressor activities. Early studies52,87 focused on IFN-α,-β and -γ and TNF-α, because they were alreadyknown to suppress a number of viruses (see above),including HIV-1 (REFS 52,88–96), when tested asreagents. However, two studies found that treatmentof CD8+ T-cell supernatants with neutralizing anti-bodies against these cytokines, or other HIV-1inhibitors, such as transforming growth factor (TGF)-βand IL-4, did not abrogate ‘CAF’ activity52,87. Thesestudies indicated that an unknown factor is responsiblefor ‘CAF’ activity, but did not eliminate the possibilitythat ‘CAF’ is a collection of known antiviral cytokines

suppresses diverse HIV-1 strains. CTL clones that arestimulated by autologous antigen-presenting cells oranti-CD3 antibodies also release a soluble non-cytolyticfactor with antiviral activity that comprises RANTES,MIP-1α, MIP-1β and unknown X4 suppressorfactors73–78. These cells release RANTES, MIP-1α andMIP-1β from their cytoplasmic granules as part of alarger glycosaminoglycan (GAG) complex75. Althoughthe immunological significance of these chemokine–GAG complexes is unclear, in the case of RANTES,MIP-1α and MIP-1β the antiviral activity is preservedafter GAG binding, whereas the receptor-activatingfunction is inhibited79. Notably, placental stromal cellswere also shown to release a factor with HIV-suppressiveactivity80. In accordance, leukaemia inhibitory factor,which is expressed in the placenta, was shown to inhibitmultiple HIV-1 strains81.

Third, the composition of the suppressor factor(s)that are released in a given system might change overtime. For example, four days after addition of antigenco-cultures of naive CD4+ T cells and dendritic cells31

secrete a factor with suppressive activity that com-prises RANTES, MIP-1α, MIP-1β and macrophage-derived chemokine (MDC), which is another HIV-suppressive chemokine82–86. The suppressive activity ofthe 4-day-old supernatant is almost entirely abrogatedby neutralizing antibodies to these chemokines31.However, culture supernatant that is collected afterstimulation is less sensitive to the neutralizing anti-chemokine antibodies. Furthermore, supernatantsthat are collected early (1 day) after addition of anti-gen suppress HIV-1 in a manner that is completelyinsensitive to these antibodies. So, the cultures releaseunknown inhibitors at certain time points and knownsuppressor factors at others. Such changes might be

DUAL-TROPIC

Also known as R5X4. An HIV-1phenotype that is defined by theuse of either CCR5 or CXCR4co-receptors for viral entry.These isolates are suppressed byRANTES, MIP-1α and MIP-1βin CCR5-dependent infectionsystems.

RANTES

MIP-1β

MIP-1α

?

?

?

Host cell

CCR5

Host cell

Bulk HIV-1supressor activity

CD8+ T cell

'CAF'

X4 or R5X4 virus(in a CCR5-negative system)

R5 or R5X4 virus(in a CCR5-dependent system)

Figure 3 | A schematic representation of the multipartite nature of the soluble HIV-1 suppressor activity produced byCD8+ T cells. RANTES, MIP-1α and MIP-1β account for the suppression of R5 viruses. Unidentified factors suppress X4 andR5X4 HIV-1 isolates in a CCR5-negative system. CAF, CD8 antiviral factor.

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sources of antiviral factors, which is necessary for proteinpurification and sequencing efforts. More recent refine-ments in genomic and microanalytical techniques havefacilitated direct examinations of primary cells forsuppressor factors. Given these new tools, a number ofadditional candidate HIV-1 suppressor factors82,101–104

have been identified in recent years (FIG. 4).Nevertheless, the identification of immunologically

relevant suppressor factors remains a tricky business.Many molecules, including substances present in com-mercial media or sera used to culture cells rather than T-cell-derived factors101, will suppress HIV-1 replicationunder certain conditions and/or at sufficient concen-trations. Therefore, it is essential to determine if acandidate factor is produced by primary cells at effec-tive antiviral concentrations. The most reliable methodis to treat conditioned media with cognate antibodiesthat either neutralize biological activity or clear thenative antigen from solution. Abrogation of HIV-1 sup-pressor activity by such treatment unambiguously showsthat the candidate factor is active at the concentrationssecreted by primary cells and therefore might be ‘relevant’to a natural immune response. Of course, such analyseswill not address the presence of redundant factors unlessan appropriate mixture of antibodies is used.

On the basis of antibody-neutralization experiments,most of the candidate human suppressor factors havebeen characterized as potentially relevant to solublesuppressor activity from at least one source (FIG. 4). Atthe same time, it is also clear that relevance is system-dependent. For example, IFN-α contributes signifi-cantly to the activity of influenza-A-stimulated PBMCs71,yet IFNs do not seem to be important components offactors with suppressor activity that are released byCD8+ T cells52,87. In the case of PBMC-derived activities,relevance is also determined by the nature of the anti-genic stimulus. So, the main suppressor factor ininfluenza-A-stimulated PBMC cultures is IFN-α71,whereas in alloantigen-stimulated PBMC cultures104 itis a heat-stable ribonuclease known as eosinophil-derived neurotoxin (EDN). This variability is perhapsnot surprising given that PBMCs can contain mixturesof cell subsets with different specificities and responseprofiles. Overall, RANTES, MIP-1α and MIP-1β providethe most overlap among systems (FIG. 4).

But the big question remains: what factors areresponsible for the CD8+ T-cell-derived suppressoractivity that is not explained by RANTES, MIP-1αand MIP-1β? Notably, the anti-chemokine antibodyexperiments (FIG. 2) indicate that these unknown factors must be significantly less potent against R5HIV-1, as CD8+ cell-culture supernatants treatedwith anti-chemokine antibodies do not exhibit resid-ual R5 suppressor activity. If these other factors wereable to suppress R5 infection, their antiviral activitiesshould have been apparent after the chemokines wereneutralized, yet this was not the case. Possibly, theunknown factors are not present in the supernatantsat sufficient concentrations to block R5 HIV-1 replica-tion. On the other hand, they might need to synergizewith one of the R5 ligands to mediate R5 suppression.

with redundant functions. Indeed, a later study showedthat a combination of antibodies to IL-10, IL-13, IFN-αand IFN-γ was able to appreciably reverse HIV-1suppressor activity in CD8+ T-cell-derived super-natants97. However, the antibody mix did not achievecomplete reversal of X4 HIV-1 suppression, indicatingthat additional unknown suppressor factors wereinvolved.

Fortunately, recent technological advances havegreatly increased the chances of successfully identifyingsoluble HIV-1 suppressor factors. The development ofherpesvirus saimiri (HVS)- and human T lymphotrophicvirus (HTLV)- immortalized T-cell lines that secrete fac-tor(s) with soluble suppressor activity53,82,98–100 has beenparticularly helpful because they provide continuous

Sensitive HIV phenotypeSuppressor factors

unidentifiedfactors

IFN-α,

CCR5 ligands

Unidentifiedfactors

Mitogen

Influenza

Whole PBMC

Stimuli

Alloantigen EDN ribonuclease,unidentifiedfactors

R5

R5

R5

X4

X4

X4

CCR5 ligands

Unidentifiedfactors

Unidentifiedfactors

Anti-CD16,cytokines

NK cellsR5

X4

R5

X4

CCR5 ligands

MDC,unidentifiedfactors

R5

X4

CCR5 ligands

Antigen anddendritic cells

Anti-CD3/CD28

CD4+ T cells

R5

X4

CCR5 ligands

Unidentifiedfactors

Antigen and antigen-presenting cells

CTL

R5

X4

CCR5 ligands

Unidentifiedprotease?

Mitogen

Anti-CD3

CD8+ T cells

Figure 4 | A variety of primary cell subsets secrete soluble HIV-1 suppressor activities inresponse to various stimuli. Candidate suppressor factors that are ‘relevant’ to these activitiesare shown. In this case, CCR5 ligands refers to RANTES, MIP-1α and MIP-1β. The HIV-1phenotype (which is defined by co-receptor preference) that has been reported to be mostsensitive to the various suppressor factors or activities is shown. CTL, cytotoxic T lymphocytes;EDN, eosinophil-derived neurotoxin; MDC, macrophage-derived chemokine; NK, natural killer;PBMC, peripheral blood mononuclear cells.

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present in their cultures were released by contaminat-ing neutrophils and not by CD8+ T cells120, and there-fore could not contribute to ‘CAF’. So, the search for aspecific CD8+ T-cell-derived HIV-1 suppressor factorthat is active against X4 viruses continues.

Although the nature of the unknown CD8-derivedfactors remains obscure, there are tantalizing indica-tions that protease activity is involved. A recent studyshowed that certain protease inhibitors abrogate thesuppressor activity in CD8+ T-cell supernatants thatact on X4 isolates121. In accordance, it was suggested thatthe HIV-suppressive fragment of bovine antithrombinIII is generated by a proteolytic activity that is greater inCD8+ T-cell supernatants from HIV-infected donorsthan from seronegative donors101. Of course, this pro-tease has no clinical relevance until it is shown to processhuman substrate into an antiviral form.

Mechanisms of non-cytolytic HIV-1 suppressionSoluble suppressor activities suppress many HIV-1strains. Such breadth of activity is consistent with thepresence of multiple suppressor mechanisms, whichcould selectively become activated according to viralphenotype and tropism (FIG. 5). In accordance with thisview, suppressor factors collectively exhibit a diverserange of antiviral mechanisms. MDC inhibits the repli-cation of R5 viruses in macrophages, but does not inter-fere in proviral DNA accumulation84. However, MDCsuppresses X4 viruses at the level of reverse transcriptionin primary T cells. The X4-suppressive effects involve aG-protein-coupled signalling pathway (C. Kleinman,A.L.D. and A. Garzino-Demo, unpublished observa-tions) that is linked either to the MDC receptor CCR4or to an unidentified receptor that might be used by anaturally truncated form of the chemokine83. EDN

Of course, these possibilities cannot be explored untilthe unknown factors have been identified.

Early on, the CXCR4 ligand SDF-1 was consideredto be a logical candidate for ‘CAF’ when it was deter-mined that it binds to CXCR4 co-receptors and blocksthe entry of both X4 and dual-tropic viruses in CCR5-minus systems63. But lymphocytes produce little or noSDF-1 and these levels do not correlate with CD8+ cell-derived suppressor activity105. Another early candidatefor ‘CAF’ was the cytokine IL-16. This cytokine is con-stitutively produced by CD4+ and CD8+ T cells106, andinhibits HIV-1 replication in vitro when tested as arecombinant molecule103,106–109. However, IL-16 is notconsidered relevant to ‘CAF’ as it was shown that theconcentrations of cytokine that are released by CD8+

cells do not correlate with levels of HIV-1 suppressoractivity in culture supernatants. Furthermore, neutral-izing anti-IL-16 antibodies do not reverse the solubleHIV-1 suppressor activities that are derived fromCD8+ cell cultures107. Other reports have suggested that‘CAF’ activity might be attributed to a fragment ofbovine antithrombin III101, or to a catalytically inactiveamino-terminal peptide fragment of urokinase-typeplasminogen activator (ATF-uPA)110,111. These possibili-ties remain to be explored. Recently, Ho and colleaguesattributed ‘CAF’ activity to the α-defensins 1, 2 and 3(REF. 97). Defensins are well known as neutrophil-derived antibiotic factors and others had alreadyobserved that they inactivate certain envelopedviruses112–114, including HIV-1 (REF. 115). However, theassertion that ‘CAF’ is explained by a combination ofRANTES, MIP-1α, MIP-1β and α−defensins102, has not‘stood the test of time’. Following evidence that theantiviral properties of ‘CAF’ and α-defensins are discor-dant116–119, the Ho group revealed that the α-defensins

Reversetranscription

Provirusintegration

LTR-driventranscription Translation Assembly Budding

EDNribonuclease

α-defensin

Attachment,fusion and

entry

IFN-α,-β IFN-α,-β,-γ

IFN-α,-β IFN-δR1IFN-δR2

IFNAR1IFNAR2

MDC (X4 viruses in PBMC)

MDC(R5 viruses inmacrophages)

Unidentified CD8+

T-cell factors

DownregulateCXCR4

ATF-uPA

Crosslink

CD4

Co-receptor

CCR5 ligands(R5 viruses)

CCR4(CCR?)

CCR4(CCR?)

CD87/u-PAR

CD4

STAT1?

STAT1?

?

?

IL-16

Figure 5 | Soluble factors might suppress HIV-1 at multiple replication steps through several mechanisms. Candidate factors and the replication steps theytarget are shown. ATF-uPa, amino-terminal peptide fragment of urokinase-type plasminogen activator; EDN, eosinophil-derived neurotoxin; IFN, interferon; IL,interleukin; LTR, long terminal repeat; MDC, macrophage-derived chemokine; STAT, signal transducer and activator of transcription; CD87/u-PAR, urokinase-typeplasminogen activator receptor.

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collective action of several factors, which might act inan indirect manner — for example, cause the release ofIFN and IL-16. It is also unclear how this mechanismwould relate to the apparent link between CD8-derivedHIV-1 suppression and protease activity (see above).

Relevance of HIV-1 suppressor factorsThe studies discussed in the previous section implythat, at least in some viral infections, soluble non-cytolytic antiviral factors might come from multiplesources, under multiple conditions and target multi-ple steps in the virus life cycle. Given this redundancy, itseems possible that soluble suppressor factors are ableto produce a barrier web that would make the averagespider blush. But is there any evidence that such a webis catching bugs in vivo? The answer to this question isfound in the clinic.

The first evidence that non-cytolytic activitiesmight be clinically relevant was provided by studiesthat showed that CD8+ T cells from asymptomaticHIV-1-infected individuals suppress HIV-1 replicationmore efficiently than cells from patients withAIDS45,46,133–135. Similarly, lymphoid tissue CD8+ T cellsfrom infected persons who remain clinically healthy forextended periods of time without clinical intervention(known as LONG-TERM NON-PROGRESSORS) were shown tomediate more potent suppression than cells from AIDSpatients135. More recent studies have focused on can-didate factors rather than gross suppressor activity.Numerous clinical studies have compared the concen-trations of RANTES, MIP-1α or MIP-1β released fromPBMCs and CD8+ T cells in vitro with the clinical statusof the cell donor136–141. These studies have repeatedlyshown a statistically significant correlation betweenincreased production of MIP-1α and/or MIP-1β fromactivated cells in vitro and favourable clinical profiles,such as increased CD4+ T-cell counts or reduced viralloads. In accordance, genetic analyses have uncoveredpolymorphisms in the human RANTES promoter thatincrease mRNA transcription and also correlate withslower HIV-1 disease progression139,142–145. Notably, arecent study found that HIV-infected patients whocontrolled their infection better after structured inter-ruption of therapy harboured R5 viruses that weresignificantly more susceptible to suppression byRANTES146.

These data support the concept that soluble non-cytolytic antiviral activities dampen HIV-1 infectionin vivo, although mainly in patients with an intrinsiccapacity to release high levels of suppressor factors.This proposal is consistent with the general model forsoluble non-cytolytic antiviral responses that is shownin FIG. 1. However, it is important to recognize thatconcentrations of CCR5 ligands and/or ‘CAF’ correlatewith disease status even when non-HIV-1 antigens ormitogens are used to stimulate the cells45,46,133–135,137–141.Furthermore, studies on CTLs derived from infectedpatients showed that the release of HIV-1 suppressorfactors is not restricted to HIV-1-specific cells77. So, theactivation of the immune system might be sufficientto suppress HIV-1 through soluble mechanisms,

ribonuclease is associated with a mechanism that blocksreplication prior to reverse transcription72,104,122. Theexact mechanism of suppression is not known, but itmight be similar to the RNase-dependent pathwaysthat are induced by IFNs6–11. IFN-α also blocks HIV-1replication prior to reverse transcription71, in part dueto the downregulation of the CXCR4 co-receptor12,13,and also at the budding and assembly steps123. ATF-uPAalso interferes with the budding and assembly steps ofHIV-1 replication through receptor-mediatedpathways110,111. The α-defensins also suppress HIV-1 byaltering the host-cell environment116, although they candirectly bind and inactivate other viruses113. In the caseof HIV-1, suppression occurs at a post-entry step thatmight be as late as proviral integration116. IL-16 is aninteresting case because it is a ligand for CD4 (REF. 106).However, this binding does not prevent virus attach-ment or gp120 interactions. Instead, CD4 crosslinkingby IL-16 homodimers or homotetramers generatesseveral second messengers that ultimately inhibit HIV-1gene transcription106,108 and, possibly, the entry of viri-ons into macrophages and dendritic cells109. Notably,IL-16 has also been shown to suppress HIV-1 replica-tion, even when present intracellularly124. As discussedabove, RANTES, MIP-1α and MIP-1β suppress theentry of R5 isolates; details of this process have beenexhaustively covered in several excellent reviews (forexample, REF. 58) and need not be described further.However, it should be noted that three other CCR5ligand — monocyte chemoattractant protein (MCP)-2 (REF. 59), LD78β60 and a natural fragment of human β-CC-chemokine (HCC)-1 (REF. 61) — also suppressR5 HIV-1 strains at the level of entry, although thesemolecules have not yet been formally linked to a solublesuppressor activity.

Crude CD8+ T-cell supernatants contain yet anotherantiviral activity that has not been assigned to anyknown factor. This activity blocks HIV-1 LONG TERMINAL

REPEAT (LTR)-driven transcription17,116,125–131, presumablythrough signal-transduction pathways that are modu-lated by receptor–ligand interactions. However, humanCD8+ cell supernatants also suppress transcription thatis driven by other retroviral promoters127, which indi-cates that the responsible antiviral agent(s) is not spe-cific to lentiviruses. In vitro assays indicate that nuclearfactor (NF)-κB, nuclear factor of activated T cells(NFAT), and STAT1 might be involved in the suppres-sive mechanism17,127,128, although suppression of a SIVmutant was observed in the absence of a NF-κB bindingdomain in the LTR131. The inhibition of LTR-driventranscription is reminiscent of HIV-1 suppression byIL-16, IFN-β, IFN-γ and TGF; however, as discussedabove, these molecules do not apparently explain theCD8-derived activity. Therefore, it is still assumed that asingle unknown factor that is released by CD8+ cellsmediates the transcriptional block. However, in thespirit of Ephraim Racker’s tenet “Do not waste cleanthinking on dirty enzymes”, caution should be exercisedwhen assigning functional mechanisms to crude mater-ial. It is possible that the antiviral effects of crude CD8+

cell medium, which is the definition of CAF, reflect the

LONG TERMINAL REPEAT

A repeated sequence, severalhundred base-pairs long, foundat the two ends of the retroviralgenome.

LONG-TERM NON-PROGRESSORS

HIV-infected persons whoremain clinically healthy for longperiods of time in the absence ofantiretroviral therapy. Thesepersons seem to have a naturalcapacity to effectively controlHIV-1 infection.

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microbial infections efficiently elicit the secretion of sol-uble HIV-1 suppressor factors at antiviral concentra-tions within reach of HIV-1 replication sites (FIG. 1).These concepts warrant further experimental evaluation.

On the other hand, there are certain clinical observa-tions that remain difficult to reconcile with the conceptof soluble suppressor activity as a mechanism ofimmunity. The infection of CD4+ T cells by HIV-1 isan example. As discussed above, naive and antigen-stimulated CD4+ subsets release soluble suppressorfactors. Yet one report suggests that the HIV-1-specificCD4+ T-cell population contains more viral DNA thanother memory CD4+ T-cell pools168. It is possible thatthe close and prolonged proximity of these cells toactively replicating HIV-1 overcomes the suppressivecapacities of soluble factors. Nevertheless, it is importantto recognize that most antigen-responsive HIV-1-specificCD4+ T cells are not infected by HIV-1 (REF. 168), despitetheir increased chances of exposure to virus. This leavesthe possibility that soluble factors provide resistance toa fraction of cells under certain conditions. Likewise, asubset of CD3– CD56+ CD4+ NK cells is persistentlyinfected with HIV-1 in vivo even though bulk CD56+

NK cells release soluble suppressor factors, includingRANTES, MIP-1α and MIP-1β169. However, other NK-cell subsets might be protected by soluble suppres-sor activity given the resistance of the bulk NK cell pop-ulation to HIV-1 infection in vitro35–37. In other words,NK cells might represent a case of selective resistance toinfection according to viral phenotype. In any case, itseems clear that the impact of soluble suppressor factorsin vitro will depend on a variety of parameters, includ-ing duration and proximity to infection, viral replica-tion kinetics versus the kinetics of production anduptake, and local viral phenotype.

Practical applications of suppressor factorsIn view of the available evidence, it is logical to considersoluble non-cytolytic suppressor activity as a modelfor developing therapeutic and prophylactic antiviralagents. Some measure of support for this proposal hasbeen provided by HBV vaccine studies carried out inboth mice and humans. DNA-based HBV vaccines wereshown to control viral replication in HBV-transgenicmice through the release of soluble non-cytolytic factorsfrom CD4+ and CD8+ lymphocytes170–172. Althoughtherapeutic HBV vaccination of humans has not pro-vided significant clinical benefit, in some studies it wasassociated with reduced serum HBV DNA levels andenhanced T-cell proliferative responses that producedhigh concentrations of TNF-α and IFN-γ171,172. Thedirect administration of IFN-α is a successful treatmentfor HCV infection.

In the case of HIV-1 infection, the arguments forexploiting soluble suppressor factors are obvious. Thefactors are non-toxic to target cells and, unlike anti-bodies or CTLs, they suppress diverse HIV-1 strains.Moreover, a significant portion of the bulk antiviralactivity of primary cells is derived from factors thatsuppress HIV-1 as a consequence of normal physiolog-ical functions. So, RANTES, MIP-1α and MIP-1β

particularly in patients that naturally release increasedconcentrations of suppressor factors. Of course, themagnitude of this ‘bystander’ suppressive effect woulddepend on the nature of the antigenic stimulus andthe frequencies of cognate responder cells that releasesuppressor factors. The ratio of naive to memory cellsmight also have an effect as memory cells shouldrapidly release soluble suppressor factors by degranu-lation, whereas naive cells produce them more slowlyde novo.

The available data further indicates that an intrinsiccapacity to secrete high concentrations of HIV-1suppressor factors might afford a certain level ofresistance to primary HIV-1 infection. Lymphocytesfrom HIV-1 seronegative people produce variablelevels of soluble HIV-1 suppressor activity followingstimulation with mitogen or other non-HIV-1 anti-gens31,47,71,72,98,104,122,140,141,147–155. Adult individuals withlymphocytes that release abnormally high concentra-tions of RANTES, MIP-1α or MIP-1β seem to remainuninfected despite repeated exposure to HIV-1 (REFS

150,151). Uninfected children born to HIV-1-positivemothers exhibited the same trait152. This intrinsiccapacity might be genetic or acquired owing to previousimmunological perturbations.

There is also evidence that certain microorganismselicit immune responses that dampen clinical HIV-1infection via soluble non-cytolytic suppressor factors.The situation seems analogous to what has beenreported for HBV suppression during LCMV infectionin transgenic mice41 or acute HAV infection inhumans42,43. Some individuals who are simultaneouslyinfected with HIV-1 and either dengue157, Orientiatsutsugamushi (the causative agent of scrub typhus)157–159,hepatitis G/GB virus C160–164 and measles morbillivirus165

have reduced HIV-1 viral loads and/or increased CD4+

T-cell counts compared with patients who are infectedonly with HIV-1 or with HIV-1 and other pathogens.As passive transfer of cell-free plasma from donorswith mild O. tsutsugamushi infection has been shownto reduce viral loads in HIV-1-infected recipients andsuppress HIV-1 replication in vitro158, a soluble factoris responsible for the effect. The activity has not beenlinked to HIV-reactive antibodies. Furthermore, invitro stimulation of PBMCs with O. tsutsugamushiinduced the production of RANTES and generatedresistance to R5, but not X4 or R5X4, HIV-1 infec-tion159. In the case of hepatitis G virus (HGV), bindingof the serum HGV E2 protein to CD81 led to increasedRANTES secretion and reduced CCR5 expression164.Notably, both dengue virus and HGV are flavivirusesand therefore might induce the release of suppressorfactors through similar immunological mechanisms. Aprospective study of commercial sex workers in Senegalshowed that HIV-2 infection reduces the risk of HIV-1infection166. Stimulated cells from these personssecreted abnormally high concentrations of RANTES,MIP-1α and MIP-1β and resisted infection by R5 HIV-1strains166). In vitro studies indicate that HTLV-II infec-tion might upregulate the production of MIP-1α167.Given these findings, it can be envisioned that some

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(to prevent spontaneous recurrent abortion) caused cellsto upregulate the release of CD8-derived soluble sup-pressor factors and CCR5 ligands. In addition, cells fromthese women became less susceptible to infection byHIV-1 and exhibited lower frequencies of co-receptorexpression185. These provocative results indicate that aprophylactic level of soluble factor release might beachieved with a vaccine that does not necessarily containHIV-1 antigens.

Concluding remarksViruses have evolved replication processes that allowthem to coexist with the immune systems of theirhosts. So, no single viral system can be used to fullydefine ‘antiviral immunity’. It is only through studiesof different viral systems that we can fully appreciatethe capacity of the immune system to mediate antivi-ral immunity. Research on primate lentiretrovirusesand other types of viruses, such as HBV, have indi-cated that soluble non-cytolytic activity might pro-vide an important mechanism for controlling at leastsome viral infections. Yet it is also possible that thenon-cytolytic suppression of antigen productionmight provide a virus with a method to persist in itshost. Indeed, some viruses might have evolved tocoexist with such suppression as it does not immedi-ately kill the virus or the host. On the other hand,there are likely to be some viral infections that do notinvolve soluble non-cytolytic factors at all.

Nevertheless, it is entirely reasonable to view thesoluble non-cytolytic antiviral activity as a promisingbasis for developing treatments for viral infections. Inthe case of HIV-1 infection, it has become evident thatsoluble suppressor activity is mediated by diversearrays of molecules that are produced by a variety ofcell subsets. This reality may not be as captivating asthe concept of a single ‘magic bullet’ factor that sup-presses HIV-1 in multiple settings, but the possibilitiesthat are provided by this diversity significantly expandthe potential for developing therapeutic or prophyl-actic antiviral strategies based on the mechanismsrevealed by soluble, non-cytolytic immunity. Thispotential will be realized and expanded as more sup-pressor factors are identified, characterized in thecontext of natural immune responses and theirmechanism of viral suppression unravelled.

become highly selective antiviral agents against R5HIV-1 strains as a consequence of their roles in animmune response.

One approach towards exploiting these features is todesign antiretroviral drugs that mimic the activities ofsoluble suppressor factors. Indeed, entry inhibitors thattarget CCR5 are likely to represent the most promisingcategory of new antiretroviral agents. Another approachmight be to deliberately modulate the immune systemto enhance the release of soluble suppressor factors fromeffector cells.A recent study showed that the bacterial G1cytostatic agent rapamycin causes PBMCs to secreteincreased concentrations of RANTES, MIP-1α andMIP-1β173 and to downregulate the CCR5 co-receptor.This effect rendered PBMCs and macrophages resistantto infection by R5 strains. Similarly, it was shown thatcells treated with other agents that inhibit the G1 phaseof the cell cycle, such as hydroxyurea, secreted increasedconcentrations of soluble HIV-1 suppressor factors, par-ticularly RANTES, MIP-1α and MIP-1β174. Rapamycinhas been used in humans to treat transplant rejectionand hydroxyurea has been tested in clinical trials incombination with anti-HIV-1 drugs. Therefore, thesecompounds might provide a feasible and expedientmeans to clinically evaluate this concept. Furthermore,these agents might be used in combination withCCR5-targeted drugs to produce a potent, synergisticantiviral effect.

Going a step further, a few research groups, includingour own, have proposed the concept of using HIV-1vaccines to enhance the capacity of the immune systemto release soluble HIV-1 suppressor activity togetherwith classical immune responses. Fortunately, primatemodels can be used to evaluate this concept as CD8+

T cells from chimpanzees, baboons and macaquessecrete a primate equivalent of ‘CAF’ that suppressesHIV-1, HIV-2 and SIV, respectively175–177. So far, pri-mate vaccine strategies that are based on non-patho-genic strains, live attenuated viruses or SIV antigensdelivered to iliac lymph nodes have associated protec-tion from virus challenge with the enhanced cellularcapacity to release CD8-derived soluble suppressor fac-tors, particularly RANTES, MIP-1α and MIP-1β38,178–184.The aim is to translate these findings into a vaccinestrategy that might be used in humans. Lehner and co-workers showed that allo-immunization of women

1. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. &Salazar-Mather, T. P. Natural killer cells in antiviral defense:function and regulation by innate cytokines. Annu. Rev.Immunol. 17, 189–220 (1999).

2. Biron, C. A. & Brossay, L. NK cells and NKT cells in innatedefence against viral infections. Curr. Opin. Immunol. 13,458–464 (2001).

3. Wallace, M., Malkovsky, M. & Carding, S. R. γ/δ Tlymphocytes in viral infections. J. Leukoc. Biol. 58, 277–283(1995).

4. Welsh, R. M. et al. α β and γ δ T-cell networks and their rolesin natural resistance to viral infections. Immunol. Rev. 159,79–93 (1997).

5. Selin, L. K., Santolucito, P. A., Pinto, A. K., Szomolanyi-Tsuda, E. & Welsh, R. M. Innate immunity toviruses: control of vaccinia virus infection by γ δ T cells. J. Immunol. 166, 6784–6794 (2001).

6. Isaacs, A. & Lindemann, J. Virus interference I. The interferon. Proc. Natl Acad. Sci. USA 147, 258 (1957).

7. Vilcek J. & Sen, G. C. in Virology (eds Fields B. N., Knipe, D. M.& Howley, P. M.) 375 (Lippencott–Raven, Philadelphia, 1996).

8. Kalvakolanu, D. V. & Borden, E. C. An overview of theinterferon system: signal transduction and mechanism ofaction. Cancer Invest. 14, 25–53 (1996).

9. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. &Schreiber, R. D. How cells respond to interferons.Annu. Rev. Biochem. 67. 227–264 (1998).

10. Guidotti, L. G. & Chisari, F. V. Noncytolytic control of viralinfections by the innate and adaptive immune response.Annu. Rev. Immunol. 19, 65–91 (2001).Excellent review of the role of soluble virussuppressor factors in HBV and other viral infections.

11. Katze, M. G., He, Y. & Gale, M. Viruses and interferon: a fightfor supremacy. Nature Rev. Immunol. 2, 675–687 (2002).

12. Shirazi, Y. & Pitha, P. M. Intereferon α-mediated inhibition ofhuman immunodeficiency virus type 1 provirus synthesis inT-cells. Virology 193, 303–312 (1993).

13. Shirazi, Y. & Pitha, P. M. Interferon downregulates CXCR4(fusin) gene expression in peripheral blood mononuclearcells. J. Human. Virol. 2, 69–76 (1998).

14. Guidotti, L. G, Guilhot, S. & Chisari, F. V. Interleukin 2 andinterferon α/β negatively regulates hepatitis B virus geneexpression in vivo by tumor necrosis factor dependentand independent pathways. J. Virol. 68, 1265–1270(1994).

15. Gilles, P. N., Fey, G. & Chisari, F. V. Tumor necrosis factor-αnegatively regulates hepatitis B virus gene expression intransgenic mice. J. Virol. 66, 3955–3960 (1992).

16. Herbein, G. & O’Brien, W. A. Tumor necrosis factor (TNF)-αand TNF receptors in viral pathogenesis Proc. Soc. Exp.Biol. Med. 223, 241–257 (2000).

NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | MAY 2004 | 411

R E V I E W S

17. Chang, T. L.-Y., Mosoian, A., Pine, R., Klotman, M. E. &Moore, J. P. A soluble factor(s) secreted from CD8+ Tlymphocytes inhibits human immunodeficiency virus type 1replication through STAT1 activation. J. Virol. 76, 569–581(2002).

18. Patterson, J. B., Thomis, D. C., Hans, S. L. & Samuel, C. E. Mechanism of interferon action: doublestranded RNA-specific adenosine deaminase from humancells is inducible by α and γ interferons. Virology 210,508–511 (1995).

19. Bovolenta, C. et al. A selective defect of IFN-γ- but not ofIFN-α-induced JAK/STAT pathway in a subset of U937clones prevents the antiretroviral effect of IFN-γ against HIV-1. J. Immunol. 162, 323–330 (1999).

20. Guidotti, L. G. et al. Cytotoxic T lymphocytes inhibit hepatitisB virus gene expression by a noncytolytic mechanism intransgenic mice. Proc. Natl Acad. Sci. USA 91, 3764–3768(1994).

21. Guidotti, L. G. et al. Intracellular inactivation of thehepatitis B by cytotoxic T lymphocytes. Immunity 4,25–36 (1996).

22. Guidotti, L. G. The role of cytotoxic T cells and cytokines inthe control of hepatitis B virus infection. Vaccine 4, 80–82(2002).

23. Nakamoto, Y., Guidotti, L. G., Pasquetto, V., Schreiber, R. D.& Chisari, F. V. Differential target cell sensitivity to cytotoxic T lymphocyte-activated death pathways in vivo. J. Immunol.158, 5692–5697 (1997).

24. Binder, D. & Kundig T. Antiviral protection by CD8+ versusCD4+ T cells: CD8+ T cells correlating with cytotoxic activityin vitro are more effective in antivaccinia virus protection thanCD4-dependent interleukins. J. Immunol. 146, 4301–4307(1991).

25. Eichelberger, M., Allan, W., Zijlstra, M., Jaenisch, R. &Doherty, P. C. Clearance of influenza virus respiratoryinfection in mice lacking Class I major histocompatibilitycomplex-restricted CD8+ T cells. J. Exp. Med. 174,875–880 (1991).

26. Kundig, T. M., Hengartner, H. & Zinkernagel R. M. T-celldependent interferon γ exerts an antiviral effect in centralnervous system but not in peripheral solid organs. J. Immunol. 150, 2316–2321 (1992).

27. Scherle, P. A., Palladino, G. & Gerhard, W. Mice can recoverfrom pulmonary influenza virus infection in the absence ofclass I-restricted cytotoxic T cells. J. Immunol. 148,212–217 (1992).

28. Spriggs, M. K. et al. β2-microglobulin-, CD8+ T-cell-deficientmice survive inoculation with hogh doses of vaccinia virusand exhibit altered IgG responses Proc. Natl Acad. Sci. USA89, 6070–6074 (1992).

29. Franco, A., Guidotti, L. G., Hobbs, M. V., Pasquetto, V. &Chisari, F. V. Pathogenic effector function of CD4+ T-helper-1cells in hepatitis B virus transgenic mice. J. Immunol. 159,2001–2008 (1997).

30. Maloy, K. J. et al. CD4+ T-cell subsets during virus infection.Protective capacity depends on effector cytokine secretionand on migratory capability. J. Exp. Med. 191, 2159–2170(2000).

31. Abdelwahab, S. F. et al. HIV-1-suppressive factors aresecreted by CD4+ T cells during primary immune responses. Proc. Natl Acad. Sci. USA 100, 15006–15010(2003).

32. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. &Salazar-Mather, T. P. Natural killer cells in antiviral defense:function and regulation by innate cytokines. Annu. Rev.Immunol. 17, 189–220 (1999).

33. Orange, J. S., Wang, B., Terhorst, C. & Biron, C. A.Requirement for natural killer cell-produced interferon γ indefense against murine cytomegalovirus infection andenhancement in defense pathway by interleukin 12administration. J. Exp. Med. 182, 1045–1056 (1995).

34. Orange, J. S. & Biron, C. A. Characterization of early IL-12,IFN-α/β, and TNF effects on antiviral state and NKresponses during murine cytomegalovirus infection. J. Immunol. 156, 4746–4756 (1996).

35. Fehniger, T. A. et al. Natural killer cells from HIV-1+ patientsproduce C-C chemokines and inhibit HIV-1 infection. J. Immunol. 161, 6433–6438 (1998).

36. Oliva, A. et al. Natural killer cells from humanimmunodeficiency virus (HIV)-infected individuals are animportant source of CC-chemokines and suppress HIV-1entry and replication in vitro. J. Clin. Invest. 102, 223–231(1998).

37. Kottilil, S. et al. Innate immunity in human immunodeficiencyvirus infection: effect of viremia on natural killer cell function.J. Infect. Dis. 187, 1038–1045 (2003).

38. Lehner, T. et al. The role of γ/δ T cells in generating antiviralfactors and β-chemokines in protection against mucosalsimian immunodeficiency virus infection. Eur. J. Immunol.30, 2245–2256 (2000).

39. Lukacher A. E., Braciale, V. L. & Braciale, T. J. In vivoeffector function of influenza virus-specific cytotoxic Tlymphocyte clones is highly specific. J. Exp. Med. 160,814–825 (1984).

40. McIntyre, K. W., Bukowski, J. F. & Welsh, R. M. Exquisitespecificity of adoptive immunization in arenavirus-infectedmice. Antiviral Res. 5, 299–305 (1985).

41. Guidotti, L. G. et al. Viral cross talk: intracellular inactivationof the hepatitis B virus during an unrelated infection of theliver. Proc. Natl Acad. Sci. USA 93, 4589–4594 (1996).

42. Tassopoulos, N. et al. Double infections with hepatitis A andB viruses. Liver 5, 348–353 (1985).

43. Van Nunen, A. B., Pontesilli, O., Uytdehaag, F., Osterhaus,A. D. & de Man, R. A. Suppression of hepatitis B virusreplication mediated by hepatitis A-induced cytokineproduction. Liver 21, 45–49. (2001).

44. Walker, C. M., Moody, D. J., Stites, D. P. & Levy, J. A. CD8+ lymphocytes can control HIV infection in vitro bysuppressing virus replication. Science 234, 1563–1566(1986).Provides the first description of soluble HIVsuppressor activity released by CD8+ T cells. This activity was later called CAF.

45. Mackewicz, C. E. & Levy, J. A. CD8+ cell anti-HIV activity:noncytolytic suppression of virus replication. AIDS Res.Hum. Retrovir. 8, 1039–1050 (1992).

46. Levy, J. A., Mackewicz, C. E. & Barker, E. Controlling HIVpathogenesis: the role of the noncytotoxic anti-HIV responseof CD8+ T cells. Immunol. Today 17, 217–224 (1996).

47. Barker, T. D., Weissman, D., Daucher, J. A., Roche, K. M. & Fauci A. S. Identification of multiple and distinct CD8+ Tcell suppressor activities: dichotomy between infected and uninfected individuals, evolution with progression ofdisease, and sensitivity to γ irradiation. J. Immunol. 156,4476–4483 (1996)

48. Wiviott, L. D., Walker, C. M. & Levy, J. A. CD8+ lymphocytessuppress HIV production by autologous CD4+ cells withouteliminating the infected cells from culture. Cell. Immunol.128, 628–634 (1990).

49. Walker, C. M., Erickson, A. L., Hsueh, F. C. & Levy, J. A.Inhibition of human immunodeficiency virus replication inacutely infected CD4+ cells by CD8+ cells involves anoncytotoxic mechanism. J. Virol. 65, 5921–5927(1991).

50. Walker, C. M. & Levy, J. A. A diffusible lymphokine producedby CD8+ T lymphocytes suppresses HIV replication.Immunology 66, 628–630 (1989).

51. Brinchmann, J. E., Gaudernack, G. & Vartdal, F. CD8+ T cells inhibit HIV replication in naturally infected CD4+ T cells. J. Immunol. 144, 2961–2966 (1990).

52. Mackewicz, C. E., Ortega, H. W. & Levy, J. A. Effect ofcytokines on HIV replication in CD4+ lymphocytes: lack ofidentity with the CD8+ cell antiviral factor. Cell. Immunol.153, 329–343 (1994).Provides evidence that the antiviral effect of CAF wasnot entirely due to any of the non-cytolytic antiviralcytokines known at the time.

53. Cocchi, F. et al. Identification of RANTES, MIP-1α, and MIP-1β as the major HIV-suppressive factors produced byCD8+ T cells. Science 270,1811–1815 (1995).The first report to identify a new HIV suppressor factorand reveal that certain chemokines are componentsof CAF. The paper also showed that solublesuppressor activities are composed of multiplecomponents.

54. Alkhatib, G. et al. CC CKR5: a RANTES, MIP-1α, MIP-1βreceptor as a fusion cofactor for macrophage-tropic HIV-1.Science 272,1955–1958 (1996).

55. Dragic, T. et al. HIV-1 entry into CD4 1 cells is mediated bythe chemokine receptor CC-CKR–5. Nature 381,667–673(1996).

56. Choe, H. et al. The β-chemokine receptors CCR3 andCCR5 facilitate infection by primary HIV-1 isolates. Cell 85,1135–1148 (1996).

57. Deng, H. K. et al. Identification of a major co-receptor forprimary isolates of HIV-1. Nature 381, 661–666 (1996).

58. Berger, E. A., Murphy, P. M. & Farber, J. M. Chemokinereceptors as HIV-1 coreceptors: roles in viral entry, tropism,and disease. Annu. Rev. Immunol. 17, 657–700 (1999).

59. Gong, W. et al. Monocyte chemotactic protein-2 activatesCCR5 and blocks CD4/CCR5-mediated HIV-1entry/replication. J. Biol. Chem. 273, 4289–4292 (1998).

60. Bertini, R. et al. Identification of MIP-1α/LD78 as amonocyte chemoattractant released by the HTLV-I-transformed cell line MT4. AIDS Res Hum Retrovir. 11,155–160 (1995).

61. Detheux, M. et al. Natural proteolytic processing ofhemofiltrate CC chemokine 1 generates a potent CCchemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties. J. Exp. Med. 192, 1501–1508 (2000).

62. Feng, Y., Broder, C. C., Kennedy, P. E. & Berger, E. A. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G-protein coupled receptor. Science 272,872–877 (1996).This seminal paper demonstrates that certainchemokine receptors act as entry co-receptors for HIV-1.

63. Bleul, C. C. et al.The lymphocyte chemoattractant SDF-1 isa ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382,829–833 (1996).

64. Mackewicz, C. E., Craik, C. S., & Levy, J. A. The CD8+ cellnoncytotoxic anti-HIV response can be blocked byprotease inhibitors. Proc. Natl Acad. Sci. USA 100,3433–3438 (2003).

65. Mackewicz, C. E. et al. Lack of the CD8+ cell anti-HIV factorin CD8+ cell granules. Blood 102, 180–183 (2003).

66. Mengozzi, M. et al. Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication in memory CD4 T cells.Proc. Natl Acad. Sci. USA 98,11644–11649 (2001).

67. Levine, B. L. et al. Antiviral effect and ex vivo CD4+ T cellproliferation in HIV-positive patients as a result of CD28costimulation. Science 272, 1939–1943 (1996).

68. Riley, J. L. et al. Intrinsic resistance to T cell infection with HIV type 1 induced by CD28 costimulation. J. Immunol.158, 5545–5553 (1997).

69. Poccia, F. et al. Phospohoantigen-reactive Vγ9δ2 T lymphocytes suppress in vitro human immunodeficiencyvirus type 1 replication by cell-released antiviral factorsincluding CC chemokines. J. Infect. Dis. 180, 858–861(1999).

70. Furci, L. et al. Antigen-driven C-C chemokine-mediated HIV-1 suppression by CD4+ T cells from exposed uninfectedindividuals expressing wild-type CCR-5 allele. J. Exp. Med.186, 455–460 (1997).

71. Pinto, L. A. et al.Inhibition of human immunodeficiency virustype 1 replication prior to reverse transcription by influenzavirus stimulation. J. Virol. 74,4505–4511 (2000).Identifies IFN as an important component of thesoluble HIV-1 suppressor factor that is releasedfollowing influenza virus stimulation.

72. Pinto, L. A., Sharpe, S., Cohen, D. I. & Shearer, G. M.Alloantigen-stimulated anti-HIV activity. Blood 92,3346–3354 (1998).

73. Roso, J. F. et al. Oligoclonal CD8 lymphocytes from personswith asymptomatic human immunodeficiency virus (HIV)type 1 infection inhibit HIV-1 replication. J. Infect. Dis. 172,964–973 (1995).

74. Yang, O. O. et al. Suppression of human immunodeficiencyvirus type 1 replication by CD8+ cells: evidence for HLAclass I-restricted triggering of cytolytic and noncytolyticmechanisms. J. Virol. 71, 3120–3128 (1997).

75. Wagner, L. et al. β-chemokines are released from HIV-1-specific cytolytic T-cell granules complexed toproteoglycans. Nature 391, 908–911 (1998).

76. Price, D. A. et al. Antigen-specific release of β-chemokinesby anti-HIV-1 cytotoxic T lymphocytes. Curr. Biol. 8,355–358 (1998).

77. Van Baalen, C. A. et al. Kinetics of antiviral activity by humanimmunodeficiency virus type 1-specific cytotoxic Tlymphocytes (CTL) and rapid selection of CTL escape virusin vitro. J. Virol. 72, 6851–6857 (1998).

78. Le Borgne, S., Fevrier, M., Callebaut, C., Lee, S. P. & Riviere, Y.CD8-cell antiviral factor activity is not restricted to humanimmunodeficiency virus (HIV)-specific T cells and can blockHIV replication after initiation of reverse transcription. J. Virol.74, 4456–4464 (2000).Demonstrates that CTLs against non-HIV antigensalso release active concentrations of non-cytolyticHIV-1 suppressor factors.

79. Burns, J. M., Lewis, G. K. & DeVico, A. L. Solublecomplexes of regulated upon activation, normal T cellsexpressed and secreted (RANTES) and glycosaminoglycanssuppress HIV-1 infection but do not induce Ca2+ signaling.Proc. Natl Acad. Sci. USA 96, 14499–14504 (1999).

80. Sharma, U. K. et al. A novel factor produced by placentalcells with activity against HIV-1. J. Immunol. 161,6404–6412 (1998).

81. Patterson, B. K. et al. Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae fromnontransmitting women. J. Clin. Invest. 107, 287–294 (2001).

82. Pal, R. et al. Inhibition of HIV-1 infection by the β-chemokineMDC. Science 278, 695–698 (1997).

83. Struyf, S. et al. Enhanced anti-HIV-1 activity and alteredchemotactic potency of NH2-terminally processedmacrophage-derived chemokine (MDC) imply an additionalMDC receptor. J. Immunol. 161, 2672–2675 (1998).

84. Cota, M. et al. Selective inhibition of HIV replication inprimary macrophages but not T lymphocytes bymacrophage-derived chemokine. Proc. Natl Acad. Sci. USA97, 9162–9167 (2000).

412 | MAY 2004 | VOLUME 2 www.nature.com/reviews/micro

R E V I E W S

85. Agrawal, L., Vanhorn-Ali, Z. & Alkhatib, G. Multipledeterminants are involved in HIV coreceptor use asdemonstrated by CCR4/CCL22 interaction in peripheralblood mononuclear cells (PBMCs). J. Leukoc. Biol. 72,1063–1074 (2002).

86. Moriuchi, H. & Moriuchi, M. Dichotomous effects ofmacrophage-derived chemokine on HIV infection. AIDS 28,994–996 (1999).

87. Brinchmann J. E., Gaudernack G. & Vartdal F. In vitroreplication of HIV-1 in naturally infected CD4+ T cells isinhibited by rIFN α2 and by a soluble factor secreted byactivated CD8+ T cells, but not by rIFNβ, rIFNγ, orrecombinant tumor necrosis factor-α. J. Acquir. ImmuneDefic. Syndr. 4, 480–488 (1991).

88. Yamamoto, J. K., Barre-Sinoussi, F., Bolton V., Pedersen, N. C.& Gardner, M. B. Human α- and β-interferon but not γ- suppress the in vitro replication of LAV, HTLV-III, and ARV-2. J. Interferon Res. 6, 143–152 (1986).

89. Hammer, S. M., Gillis, J. M., Groopman, J. E. & Rose, R. M.In vitro modification of human immunodeficiency virusinfection by granulocyte-macrophage colony-stimulatingfactor and γ interferon. Proc. Natl Acad. Sci. USA 83,8734–8738 (1986).

90. Nakashima, H., Yoshida, T., Harada, S, & Yamamoto, H.Recombinant human interferon γ suppresses HTLV-IIIreplication in vitro. Int. J. Cancer 38, 433–436 (1986).

91. Poli, G., Orenstein, J. M., Kinter, A., Folks, T. M. & Fauci, A. S.Interferon-α but not AZT suppresses HIV expression inchronically infected cell lines. Science 244, 575–577 (1989).

92. Wong, G. H., Krwoka, J. F., Stites, D. P. & Goeddel, D. V. In vitro anti-human immunodeficiency virus activities oftumor necrosis factor-α and interferon-γ. J. Immunol. 40,120–124 (1988).

93. Williams, G. J. & Colby, C. B. Recombinant humaninterferon-β suppresses the replication of HIV and actssynergistically with AZT. J. Interferon Res. 9, 709–718 (1989).

94. Gendelman, H. E. et al. Regulation of HIV replication ininfected monocytes by IFN-α. Mechanisms for viralrestriction. J. Immunol. 145, 2669–2676 (1990).

95. Hartshorn, K. L., Neumeyer, D., Vogt, M. W., Schooley, R. T.& Hirsch, M. S. Activity of interferons α, β, and γ againsthuman immunodeficiency virus replication in vitro. AIDS Res. Hum. Retrovir. 3, 125–133 (1987).

96. Kornbluth, R. S., Oh, P. S., Munis, J. R., Cleveland, P. H. &Richman, D. D. The role of interferons in the control of HIVreplication in macrophages. Clin. Immunol. Immunopathol.54, 200–219 (1990).

97. Moriuchi, H., Moriuchi, M., Combadiere, C., Murphy P. M. &Fauci, A. S. CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppressHIV-1 replication in monocyte/macrophages. Proc. NatlAcad. Sci. USA. 93, 15341–15345 (1996).

98. Saha, K., Bentsman, G., Chess, L. & Volsky, D. J.Endogenous production of β-chemokines by CD4+, but not CD8+, T-cell clones correlates with the clinical state ofhuman immunodeficiency virus type 1 (HIV-1)-infectedindividuals and may be responsible for blocking infectionwith non-syncytium-inducing HIV-1 in vitro. J. Virol. 72,876–881 (1998).

99. Leith, J. G. et al. T cell-derived suppressive activity: evidence ofautocrine noncytolytic control of HIV type 1 transcription andreplication. AIDS Res. Hum. Retrovir. 15, 1553–1561 (1999).

100. Mosoian, A., Teixeira, A., Caron, E., Piwoz, J. & Klotman, M. E.CD8 cell lines isolated from HIV-1-infected children havepotent soluble HIV-1 inhibitory activity that differs from β-chemokines. Viral Immunol. 13, 481–495 (2000).

101. Geiben-Lynn, R., Brown, N., Walker, B. D. & Luster, A. D.Purification of a modified form of bovine antithrombin III asan HIV-1 CD8+ T-cell antiviral factor. J. Biol. Chem. 277,42352–42357 (2002).Demonstrates that components of commercialmedia or serum additives (perhaps generated bycellular proteases) might contribute to soluble HIV-suppressor activities.

102. Zhang, L. et al. Contribution of human defensin 1, 2,and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science 298,995–1000 (2002).

103. Baier, M., Werner, A., Bannert, N., Metzner, K. & Kurth, R.HIV suppression by interleukin-16. Nature 378, 563 (1995).

104. Rugeles, M. T. et al. Ribonuclease is partly responsible forthe HIV-1 inhibitory effect activated by HLA alloantigenrecognition. AIDS 17, 481–486 (2003).Identifies EDN ribonuclease as an important solubleHIV-1 suppressor factor that is released byalloantigen-stimulated cells.

105. Lacey, S., McDanal, C. B., Horuk, R. & Greenberg, M. L.The CXC chemokine stromal cell derived factor 1 is notresponsible for CD8+ T cell suppression of syncytia-indicingstrains of HIV-1. Proc. Natl Acad. Sci. USA 94, 9842–9847(1997).

106. Cruikshank, W. W., Kornfeld, H., & Center, D. M. Interleukin-16. J. Leukoc Biol. 67, 757–766 (2000).

107. Mackewicz, C. E., Levy J. A., Cruikshank, W. W., Kornfeld, H.& Center, D. M. Role of IL-16 in HIV replication. Nature 383, 488–489 (1995).

108. Maciaszek, J. W. et al. IL-16 represses HIV-1 promoteractivity. J. Immunol. 158, 5–8 (1997).

109. Truong, M. et al. Interleukin-16 inhibits humanimmunodeficiency virus type 1 entry and replication inmacrophages and dendritic cells. J. Virol. 73, 7008–7013(1999).

110. Wada, M. et al. Amino-terminal fragment of urokinase-typeplasminogen activator inhibits HIV-1 replication. Biochem.Biophys. Res. Commun. 284, 346–351 (2001).

111. Alfano, M., Sidenius, N., Panzeri, B., Blasi, F. & Poli G.Urokinase-urokinase receptor interaction mediates aninhibitory signal for HIV-1 replication. Proc. Natl Acad. Sci.USA 99, 8862–8867 (2002).

112. Sinha, S., Cheshenko, N., Lehrer, R. I. & Herold, B. C. NP-1,a rabbit α-defensin, prevents the entry and intercellularspread of herpes simplex virus type 2. Antimicrob. AgentsChemother. 47, 494–500 (2003).

113. Daher, K. A., Selsted, M. E. & Lehrer, R. I. Direct inactivationof viruses by human granulocyte defensins. J. Virol. 60,1068–1074 (1986).

114. Bastian, A., & Schafer, H. Human α-defensin 1 (HNP-1)inhibits adenoviral infection in vitro. Regul. Pept. 101,157–161 (2001).

115. Nakashima, H., Yamamoto, N., Masuda, M. & Fujii, N.Defensins inhibit HIV replication in vitro. AIDS 7, 1129(1993).

116. Chang, T. L., Francois, F., Mosoian, A. & Klotman, M. E.CAF-mediated human immunodeficiency virus (HIV) type 1transcriptional inhibition is distinct from α-defensin-1 HIVinhibition. J. Virol. 77, 6777–6784 (2003).

117. Mackewicz, C. E. et al. Lack of the CD8+ cell anti-HIV factorin CD8+ cell granules. Blood 102, 180–183 (2003).

118. Borregaard, N. & Cowland, J. B. Granules of the humanneutrophilic polymorphonuclear leukocyte. Blood 10,3503–3521 (1997).

119. Mackewicz, C. E. et al. α-defensins can have anti-HIVactivity but are not CD8 cell anti-HIV factors. AIDS 17,23–32 (2003).

120. Zhang, L., Lopez, P., He, T., Yu, W. & Ho, D. D. Retraction ofan interpretation. Science 303, 467 (2004).

121. Mackewicz, C. E., Craik, C. S. & Levy, J. A. The CD8+ cellnoncytotoxic anti-HIV response can be blocked by proteaseinhibitors. Proc. Natl Acad. Sci. USA 100, 3433–3438 (2003).

122. Pinto, L. A., Blazevic, V., Shearer, G. M., Patterson, B. K. &Dolan, M. J. Alloantigen-induced anti-HIV activity occursprior to reverse transcription and can be generated byleukocytes from HIV-infected individuals. Blood 95,1875–1876 (2000).

123. Fernie, B. F., Poli, G. & Fauci, A. S. α-interferon suppressesvirion but not soluble human immunodeficiency virus antigenproduction in chronically infected T-lymphocytic cells. J. Virol. 65, 3968–3971 (1991).

124. Zhou, P., Goldstein, S., Devadas, K., Tewari, D. & Notkins,A. Human CD4+ cells transfected with IL-16 cDNA areresistant to HIV-1 infection: inhibition of mRNA expression.Nature Med. 3, 659–664 (1997).

125. Chen, C. H., Weinhold, K. J., Bartlett, J. A., Bolognesi, D. P.& Greenberg, M. L. CD8 T lymphocyte-mediated inhibitionof HIV-1 long terminal repeat transcription: a novel antiviralmechanism. AIDS Res. Hum. Retrovir. 9, 1079–1086 (1993).Shows that ‘CAF’ contains an unknown factor thatinhibits HIV-1 transcription.

126. Mackewicz, C. E., Blackbourn, D. J. & Levy, J. A. CD8 T cells suppress human immunodeficiency virusreplication by inhibiting viral transcription. Proc. Natl Acad.Sci. USA 92, 2308–2312 (1995).

127. Copeland, K. F., McKay, P. J. & Rosenthal, K. L.Suppression of activation of the human immunodeficiencyvirus long terminal repeat by CD8 T cells is not lentivirusspecific. AIDS Res. Hum. Retrovir. 11, 1321–1326 (1995).

128. Copeland, K. F., McKay, P. J. & Rosenthal, K. L.Suppression of the human immunodeficiency virus longterminal repeat by CD8 T cells is dependent on the NFAT-1element. AIDS Res. Hum. Retrovir. 12, 143–148 (1996).

129. Leith, J. G. et al. T cell-derived suppressive activity: evidence ofautocrine noncytolytic control of HIV type 1 transcription andreplication. AIDS Res. Hum. Retrovir. 15, 1553–1561 (1999).

130. Tomaras, G. D. et al. CD8 T cell-mediated suppressiveactivity inhibits HIV-1 after virus entry with kinetics indicatingeffects on virus gene expression. Proc. Natl Acad. Sci. USA97, 3503–3508 (2000).

131. Locher, C. P., Witt, S. A. & Levy, J. A. The nuclear factor κBand Spl binding sites do not appear to be involved in virussuppression by CD8 T lymphocytes. AIDS 15, 2455–2457(2001).

132. Walker, C. M., Moody, D. J., Stites, D. P. & Levy, J. A. CD8+

T lymphocyte control of HIV replication in cultured CD4+ cellsvaries among infected individuals. Cell. Immunol. 119,470–475 (1989).

133. Mackewicz, C. E., Ortega, H. W. & Levy, J. A. CD8+ cell anti-HIV activity correlates with the clinical state of theinfected individual. J. Clin. Invest. 87, 1462–1466 (1991).Provides early evidence that the natural capacity toproduce increased concentrations of HIV-1suppressor factors correlates with better clinicalstatus in HIV-1 infection.

134. Stanford, S. A. et al. Lack of infection in HIV-exposedindividuals is associated with a strong CD8+ cellnoncytotoxic anti-HIV response. Proc. Natl Acad. Sci. USA96, 1030–1035 (1999).

135. Blackbourn, D. J. et al. Suppression of HIV replication bylymphoid tissue CD8+ cells correlates with the clinical stateof HIV-infected individuals. Proc. Natl Acad. Sci. USA 93,13125–13130 (1996).

136. Garzino-Demo, A., DeVico A. L. & Gallo, R. C. Chemokinereceptors and chemokines in HIV infection. J. Clin. Immunol.18, 243–255 (1998).

137. Garzino-Demo, A., DeVico, A. L., Cocchi, F. & Gallo R. C. β-chemokines and protection from HIV type 1 disease. AIDS Res. Hum. Retrovir. 14 (Suppl.), S177–S184 (1998).

138. Gallo, R. C., Garzino-Demo, A. & DeVico, A. L. HIV infectionand pathogenesis: what about chemokines? J. Clin.Immunol. 19, 293–299 (1999).

139. Garzino-Demo, A., DeVico, A. L., Conant, K. E. & Gallo R. C.The role of chemokines in human immunodeficiency virusinfection. Immunol. Rev. 177, 79–87 (2000).

140. Garzino-Demo, A. et al. Spontaneous and antigen-inducedproduction of HIV-inhibitory β-chemokines are associatedwith AIDS-free status. Proc. Natl Acad. Sci. USA 96,11986–11991 (1999).

141. Cocchi, F. et al. Higher macrophage inflammatory protein(MIP)-1α and MIP-1β levels from CD8+ T cells areassociated with asymptomatic HIV-1 infection. Proc. NatlAcad. Sci. USA 97, 13812–13817 (2000).This cross-sectional study shows that a naturalcapacity to release increased concentrations of CCR5ligands on stimulation correlates with better clinicalstatus in HIV-1 infection.

142. Liu, H. et al. Polymorphism in RANTES chemokine promoteraffects HIV-1 disease progression. Proc. Natl Acad. Sci.USA 96, 4581–4585 (1999).

143. McDermott, D. H. et al. Chemokine RANTES promoterpolymorphism affects risk of both HIV infection and diseaseprogression in the multicenter AIDS cohort study. AIDS 14,2671–2678 (2000).

144. Gonzales, E. et al. Global survey of genetic variation inCCR5, RANTES, and MIP-1α: impact on the epidemiologyof the HIV-1 pandemic. Proc. Natl Acad. Sci. USA 98,51199–5204 (2001).

145. An, P. et al. Modulating influence on HIV/AIDS by interactingRANTES gene variants. Proc. Natl Acad. Sci. USA 99,10002–10007 (2002).

146. Swiss HIV Cohort Study. Human immunodeficiency virustype 1 fitness is a determining factor in viral rebound andset point in chronic infection. J. Virol. 77, 13146–13155(2003).

147. Hsueh, F. W., Walker, C. M., Blackbourn, D. J. & Levy, J. A.Suppression of HIV replication by CD8+ cell clones derivedfrom HIV-infected and uninfected individuals. Cell. Immunol.159, 271–279 (1994).

148. Rosok, B. et al. CD8+ T cells from HIV type 1-seronegativeindividuals suppress virus replication in acutely infected cells.AIDS Res. Hum Retrovir. 13, 79–85 (1997).

149. Levy, J. A., Hsueh, F., Blackbourn, D. J., Wara, D. &Weintrub, P. S. CD8 cell noncytotoxic antiviral activity inhuman immunodeficiency virus-infected and-uninfectedchildren. J. Infect. Dis. 177, 470–472 (1998).

150. Zagury, D. et al. C-C chemokines, pivotal in protectionagainst HIV type 1 infection. Proc. Natl Acad. Sci. USA 95,3857–3861 (1998).

151. Paxton, W. A. et al. Reduced HIV-1 infectability of CD4+

lymphocytes from exposed-uninfected individuals:association with low expression of CCR5 and highproduction of β-chemokines. Virology 244, 66–73 (1998).Provides early evidence that patients who remainuninfected despite repeated exposure to HIV-1 have anatural capacity to release high concentrations ofsoluble suppressor factors.

152. Wasik, T. J. et al. Protective role of β-chemokinesassociated with HIV-specific Th responses against perinatalHIV transmission. J. Immunol. 162, 4355–4364 (1999).

153. Stranford, S. A. et al. Lack of infection in HIV-exposedindividuals is associated with a strong CD8+ cellnoncytotoxic anti-HIV response. Proc. Natl Acad. Sci. USA96, 1030–1035 (1999).

NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | MAY 2004 | 413

R E V I E W S

154. Geiben-Lynn, R. et al. Noncytolytic inhibition of X4 virus bybulk CD8+ cells from human immunodeficiency virus type 1(HIV-1)-infected persons and HIV-1-specific cytotoxic Tlymphocytes is not mediated by β-chemokines. J. Virol. 75,8306–8316 (2001).

155. Skurnick, J. H. et al. Correlates of nontransmission in USwomen at high risk of human immunodeficiency virus type 1infection through sexual exposure. J. Infect. Dis. 185,428–438 (2002).

156. Watt, G., Kantipong, P. & Jongsakul, K. Decrease in humanimmunodeficiency virus type 1 load during acute denguefever. Clin. Infect. Dis. 36, 1067–1069 (2003).

157. Watt, G. et al. HIV-1 suppression during acute scrub-typhusinfection. Lancet 356, 475–479 (2000).

158. Watt, G., Kantipong, P., Jongsakul, K., de Souza, M. &Burnouf, T. Passive transfer of scrub typhus plasma to patientswith AIDS: a descriptive clinical study. Q. J. M. 94, 599–607(2001).

159. Moriuchi, M., Tamura, A. & Moriuchi, H. In vitro reactivationof human immunodeficiency virus-1 upon stimulation withscrub typhus rickettsial infection. Am. J. Trop. Med. Hyg. 68,557–561 (2003).

160. Lefrere, J. J. et al. Carriage of GB virus C/hepatitis G virusRNA is associated with a slower immunologic, virologic, andclinical progression of human immunodeficiency virus diseasein coinfected persons. J. Infect. Dis. 179, 783–789 (1999).

161. Yeo, A. E. et al. Effect of hepatitis G virus infection onprogression of HIV infection in patients with hemophilia.Multicenter hemophilia cohort study. Ann. Intern. Med. 132,959–963 (2000).

162. Tillmann, H. L. et al. Infection with GB virus C and reducedmortality among HIV-infected patients. N. Engl. J. Med. 345,715–724 (2001).

163. Tillmann, H. L. & Manns, M. P. GB virus-C infection inpatients infected with the human immunodeficiency virus.Antiviral Res. 52, 83–90 (2001).

164. Nattermann, J. et al. Regulation of CC chemokine receptor5 in hepatitis G virus infection. AIDS. 17, 1457–1462 (2003).

165. Moss, W. J. et al. Suppression of human immunodeficiencyvirus replication during acute measles. J. Infect. Dis. 185,1035–1042 (2002).

166. Kokkotou, E. G. et al. In vitro correlates of HIV-2-mediatedHIV-1 protection. Proc. Natl Acad. Sci. USA 97, 6797–6802(2000).

167. Casoli, C. et al. HTLV-II down-regulates HIV-1 replication inIL-2-stimulated primary PBMC of coinfected individualsthrough expression of MIP-1α. Blood 95, 2760–2769 (2000).

168. Douek, D. C. et al. HIV preferentially infects HIV-specificCD4+ T cells. Nature 417, 95–98 (2002).

169. Valentin, A. et al. Persistent HIV-1 infection of natural killercells in patients receiving highly active antiretroviral therapy.Proc. Natl Acad. Sci. USA 99, 7015–7020 (2002).

170. Mancini, M., Hadchouel, M., Tiollais, P. & Michel, M. L.Regulation of hepatitis B virus mRNA expression in ahepatitis B surface antigen transgenic mouse model by IFN-γ-secreting T cells after DNA-based immunization. J. Immunol. 161, 5564–5570 (1998).

171. Couillin, I. et al. Specific vaccine therapy in chronic hepatitisB: induction of T cell proliferative responses specific forenvelope antigens. J. Infect. Dis. 180, 15–26 (1999).

172. Ren, F., et al. Cytokine-dependent anti-viral role of CD4-positive T cells in therapeutic vaccination againstchronic hepatitis B viral infection. J. Med. Virol. 71,376–384 (2003).

173. Heredia, A. et al. Rapamycin causes down-regulation ofCCR5 and accumulation of anti-HIV β-chemokines: anapproach to suppress R5 strains of HIV-1. Proc. Natl Acad.Sci. USA 100, 10411–10416 (2003).

174. Heredia, A. et al. Induction of G1 cycle arrest in Tlymphocytes results in increased extracellular levels of β-chemokines: a strategy to inhibit R5 HIV-1. Proc. Natl Acad.Sci. USA 100, 4179–4184 (2003).

175. Castro, B. A., Walker, C. M., Eichberg, J. W. & Levy, J. A.Suppression of human immunodeficiency virus replicationby CD8+ cells from infected and uninfected chimpanzees.Cell. Immunol. 132, 246–255 (1991).

176. Blackbourn, D. J. et al. CD8+ cells from HIV-2-infectedbaboons control HIV replication. AIDS 11, 737–746 (1997).

177. Kannagi, M., Chalifoux, L. V., Lord, C. I. & Letvin, N. L. Sup-pression of simian immunodeficiency virus replication in vitro by CD8+ lymphocytes. J. Immunol. 140, 2237–2242(1988).

178. Gauduin, M. C., Glickman, R. L., Means, R. & Johnson, R. P.Inhibition of simian immunodeficiency virus (SIV) replicationby CD8+ T lymphocytes from macaques immunized withattenuated SIV. J. Virol. 72, 6315–6324 (1988).

179. Heeney, J. L. et al. β-chemokines and neutralizing antibodytiters correlate with sterilizing immunity generated in HIV-1vaccinated macaques. Proc. Natl Acad. Sci. USA 95,10803–10808 (1988).

180. Leno, M. et al. CD8+ lymphocyte antiviral activity in monkeysimmunized with SIV recombinant poxvirus vaccines: potentialrole in vaccine efficacy. AIDS Res. Hum. Retrovir. 15,461–470 (1999).

181. Heeney, J. et al. HIV-1 vaccine-induced immune responseswhich correlate with protection from SHIV infection:compiled preclinical efficacy datafrom trials with tendifferent HIV-1 vaccine candidates. Immunol. Lett. 66,189–195 (1999).

182. Ahmed, R. K. et al. β-chemokine production in macaquesvaccinated with live attenuated virus correlates withprotection against simian immunodeficiency virus (SIVsm)challenge. J. Gen. Virol. 80, 1569–1574 (1999).

183. Lehner, T., et al. Up-regulation of β-chemokines and down-modulation of CCR5 co-receptors inhibit simianimmunodeficiency virus transmission in non-humanprimates. Immunology 99, 569–577 (2000).Evidence from a primate infection model whichindicates that vaccines might afford protection fromHIV-1 infection by modulating the capacity of theimmune system to release soluble suppressorfactors.

184. Ahmed, R. K. et al. Spontaneous production of RANTESand antigen-specific IFN-γ production in macaquesvaccinated with SHIV-4 correlates with protectionagainst SIVsm challenge. Clin. Exp. Immunol. 129,11–18 (2002).

185. Wang, Y. et al. Allo-immunization elicits CD8+ T cell-derived chemokines, HIV suppressor factors and resistanceto HIV infection in women. Nature Med. 5, 1004–1009(1999).

Competing interests statementThe authors declare that they have no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/Entrez/hepatitis A virus | hepatitis B virus | HIV-1 | human T lymphotropicvirus | simian immunodeficiency virus | vaccinia virus | vesicularstomatitis virusLocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/CCR5 | CXCR4 | IFNs | TNFSwissProt: http://www.ca.expasy.org/sprot/eosinophil-derived neurotoxin | MIP-1α | MIP-1β

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