549

Several human inherited immune disorders lead to the samefatal lymphoproliferative syndrome, called the hemophagocyticsyndrome. Through defective perforin expression or transport,these disorders highlight the determinant role of the secretorycytotoxic pathway in the regulation of the immune responseand in lymphocyte homeostasis. In addition, new effectors ofthis secretory pathway have been identified.

AddressesInstitut National de la Santé et de la Recherche Médicale (INSERM)U429, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France*e-mail: sbasile@necker.fr†e-mail: fischer@necker.fr

Current Opinion in Immunology 2001, 13:549–554

0952-7915/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsAPC antigen-presenting cellCHS Chediak−Higashi syndromeCTL cytotoxic T lymphocyteEBV Epstein−Barr virusFasL Fas ligandFHL familial hemophagocytic lymphohistiocytosisGS Griscelli syndromeHPS hemophagocytic syndromeLCMV lymphocytic choriomeningitis virusXLP X-linked lymphoproliferative syndrome

IntroductionThe immune system has a remarkable capacity to maintain astate of dynamic equilibrium despite recurrent exposure to adiverse array of organisms and constant exposure to self-anti-gens. Two major immune cell death pathways allow theimmune response to be self-limited and decline with timeafter antigenic stimulation. One involves a nonsecretory path-way that is based on the interaction of Fas ligand (FasL) on thesurface of the T cells with the apoptosis-inducing receptor,Fas, expressed on the target cell. The cytoplasmic tail of Fascontains a motif called the ‘death domain’, which triggers adeath signal upon oligomerization and triggers the activationof the caspase cascade. The second cell death pathway relieson granule exocytosis, which involves the secretion of lyticproteins, perforin and a series of serine proteases calledgranzymes. Adhesion of the cytotoxic T lymphocytes (CTLs)to the target cell, via the interaction between the TCR and theantigen−MHC complex, triggers a Ca2+-dependent degranu-lation process in the effector cells which causes target cell lysis[1–4]. A number of studies have shown that this pathway operates in viral defense and immune surveillance against can-cer. Until recently, however, only the nonsecretory pathwaywas known to be a determinant in lymphocyte homeostasis inhumans, through its role in peripheral tolerance [5].

The recent molecular characterization of several inheritedimmune disorders that are listed in Table 1, all of which are

characterized by a defect in the secretory pathway, provides a series of observations contributing to the under-standing of this cytotoxic pathway in the control ofimmune responses. Indeed, all of these conditions share acommon phenotype — the unbalanced expansion and activation of lymphocytes (mostly CD8+), and macrophageactivation resulting in hemophagocytosis, hence its namehemophagocytic syndrome (HPS). The present review willfocus on the recent advances in the characterization of themolecular mechanisms associated with this secretory cytotoxic pathway and its role in homeostasis.

Features of hemophagocytic syndromeThe HPS observed in familial hemophagocytic lympho-histiocytosis (FHL), Chediak−Higashi syndrome (CHS),Griscelli syndrome (GS) and in X-linked lymphoprolifera-tive syndrome (XLP) is characterized by lymphoid organand extranodal infiltration by polyclonal T cells, mostly of the CD8+ subset [6]. These T cells exhibit activationmarkers (e.g. CD25 and HLA class II). Associated withactivated T cells are activated macrophages that phagocy-tose blood cells. This condition does not, in most cases,spontaneously remit. In the brain, the inflammatory cellsform perivascular foci, suggestive of a blood-derived tis-sue infiltration. Immune cell infiltration results in massivetissue necrosis, organ failure and death in the absence ofimmunosuppressive treatment. Massive cytokine releaseis another hallmark of the condition, which includes highplasma levels of IL-1, IL-6, TNF and IFN-γ [7,8]. It is thought that macrophage activation is mostly the consequence of massive TNF production leading tohemophagocytosis, high triglyceride levels and coagula-tion disorders. HPS is in most cases triggered by a viralinfection, especially of the herpes group (Epstein−Barrvirus [EBV] and cytomegalovirus). Of note, there is neither evidence of inappropriate B cell activation norautoimmune manifestations.

Human and murine genetic defects affectingthe secretory cytotoxic pathwayThe various molecular defects that have recently beenidentified as leading to HPS all disrupt the secretory cytotoxic pathway. FHL is one of these immune disorders.It is characterized by a major immune dysregulation occurring in young children that invariably leads to deathunless treated with allogenic stem cell transplantation [9].Perforin deficiency has recently been identified as anunderlying cause of FHL [10••,11] (Figure 1). As a conse-quence of gene mutations, perforin expression is barelydetected in patients’ cytotoxic granules. Consequently,T and NK cell cytotoxic activities are impaired. The per-forin defect accounts for only one third of the FHL cases.The other causes remain unidentified, one of the candi-date genes being located on chromosome 9q21.3−q22 [12].

The role of cytotoxicity in lymphocyte homeostasisGeneviève de Saint Basile* and Alain Fischer†

In CHS, a defect in a lysosomal trafficking regulator (designated CHS1 in humans and LYST in mice) [13,14],a cytosolic large protein with no clear homology with otherknown proteins [15], affects the sorting of several lysoso-mal proteins, among them the MHC class II molecules inB cells [16], the regulatory molecule CTLA-4 [17] and lyticenzymes — granzymes and perforin — present in the cytotoxic cells [18]. As a consequence of CHS1/LYSTmutation, lytic enzymes accumulate in giant intracellulargranules that may be unable to release the enzymes,accounting for the defective secretory cytotoxic activity ofT and NK cells from the patients with CHS.

In GS, mutations in the gene for the small GTPase,Rab27a, a regulator of intracellular vesicular traffic, leads todefective cytotoxic granule exocytosis [19••]. This defectalso impairs the secretory cytotoxic activity [19••]. A defectin the molecular motor protein myosin, Va — another protein involved in intracellular trafficking — can also leadto GS but, in this case, patients present partial albinismand severe neurological impairment but do not developHPS [20]. These findings demonstrate that, in contrast toRab27a, myosin Va is not an essential component of thesecretory cytotoxic pathway, which functions normally inthese patients. In CHS and GS, the intracellular traffickingdefects also occur in melanocytes, accounting for the partial albinism characteristic of these conditions (Table 1).Several lines of evidence suggest that, in melanocytes,Rab27a enables the capture of melanosomes to the periph-ery, by recruiting myosin Va molecular motor protein at thevesicle surface [21,22].

The same genetic defects have been engineered or foundin mice but, surprisingly, none of these mouse models,although bred in non-protected facilities, spontaneouslydevelops HPS. However, when challenged with certainstrains of viruses, such as the lymphocytic choriomeningi-tis virus (LCMV), perforin-knockout mice (perforin−/−

mice) develop an overeactive virus-specific T cell responseand mice die within a few weeks as a consequence of

immune damage mediated by CTLs, macrophages, IFN-γand TNF [23,24•,25••,26••]. Pathology appears to be verysimilar or identical to that of human FHL. A similar out-come has been triggered by infections of mice with Herpessimplex virus or the Theiler virus [27]. Beige and ashenmice, the murine homologue of CHS and GS, respective-ly, present a phenotype close to their human counterparts.In association with a pigmentary dilution, both mutantsdisplay a defective secretory cytotoxic function that, inashen mice, is not the consequence of defective lytic granule polarization but to an impairment of membranedocking and exocytosis [28•,29•]. Similarly to what isobserved for perforin−/− mice, these murine mutants do notdevelop spontaneous HPS and the consequences ofLCMV challenge in these mutant mice have not yet beenfinely evaluated.

Mechanisms for terminating immune responsesthat follow viral infection Recent studies have shown that the primary CD8+ T cellresponse to a viral antigen is generally characterized by amassive proliferation of antigen-specific T cells, in particular of the clones specific for the dominant antigenicepitopes [30••,31]. There is a massive generation of antigen-specific CTLs, which efficiently kill infected target cells while releasing a great quantity of mediatorssuch as IFN-γ. The latter can inhibit viral replication andtake part in the macrophage activation and inflammatoryreaction. As the levels of antigen fall, most of these T cellsdie, the few remaining cells probably constituting the poolof memory cells able to mediate responses upon furtherexposure to antigen [32].

The disappearance of this effector population is essential asits persistence causes a fatal HPS, as observed in inheritedhuman HPS or in virus-challenged perforin−/− mice.Perforin-deficient mice, unlike normal mice, are unable toclear Theiler’s virus infection and die as a consequence of anexcessive T cell activation, independently of antigen load[27]. Similarly, chronic infection of perforin-deficient mice

550 Immunogenetics

Table 1

Genetically determined diseases that display hemophagocytic syndrome.

Human disease Natural murine equivalent Phenotype Affected gene/protein

FHL Not applicable HPS PerforinFHL1 located in 9q21.3–q22

Other(s)

CHS Beige Partial albinism CHS1/LYSTGiant intracellular granulation

HPS

GS Ashen Partial albinism Rab27aHPS

XLP Purtilo EBV-induced HPS SAP/SH2D1A/DSHPAgammaglobulinemia

Lymphoma

with LCMV variants has shown that, rather than a defect inviral clearance, the persistence of the bulk of activatedCD8+ cells and the deleterious action of the cytokines theysecrete are responsible for the severity of this syndrome[24•,25••,26••,33,34]. Depletion of the CD8+ T cells orinduction of thymic tolerance to a viral epitope considerablyreduces the reactive immunopathology in these mice[23,35]. These findings indicate that passive elimination ofactivated T cells cannot solely account for re-establishmentof the homeostatic balance. Therefore, the question is raisedabout the mechanism(s) by which the immune system manages to eliminate more than 90% of the CD8+ effectorcell population expanded during an infection and thusrestore homeostasis. Analysis of the consequences of natural mutants of the secretory cytotoxic pathway showsthat it is essential to this cellular contraction.

The process of passive death through deprivation of survivalstimuli and downregulation of anti-apoptotic factor expression,such as those of the Bcl-2 family, or through the exhaustion ofthe antigen-specific clones [1] are clearly not sufficient torapidly reach the equilibrium state. Active termination path-ways through regulatory molecules such as FOXP3 (deficientin the human homologue of the scurfy mice [36••,37••]),CTLA-4 or Fas−FasL, or through the secretion of inhibitorycytokines such as IL-10 or TGF-β1 [1] can also not be consid-ered to play a major role in this process. Mice or humansgenetically deficient for these regulatory molecules develop afatal lymphoproliferative disease, but this occurs in absence ofinfections and mainly relies on CD4+ T cells. Thus these mol-ecules are instead involved in the regulation of self-tolerancein the periphery. Nevertheless, considering the ‘enhanced’phenotype of Fas−/−perforin−/− double-knockout mice,autoimmune and exogeneous responses are probably overlap-ping processes that cannot be entirely distinguished [25••].

How do cytotoxic granules regulate T cellproliferation? Several hypotheses can be proposed for cytotoxic-granuleregulation of T cell proliferation, involving cis/trans processesand different target cells. The assumption of a cis-action (sui-cide) of the secretory cytotoxic pathway following the killingprocess of the target cells is unlikely, taking into account thelow sensitivity of CTLs to their own lytic enzymes. However,it remains possible that at some stage or after a certain num-ber of divisions, CTLs become sensitive to the action ofsecreted perforin. Interestingly, it is worth noting that the regulatory effects of perforin are better detected under condi-tions of chronic TCR stimulation [25••,26••]. A trans-actionmechanism of cytotoxic granules is strongly supported by theobservation that, following allogenic stem cell transplantation,the presence of even a few donor cells in the recipient is usually sufficient to control FHL, CHS and GS durably [38].Trans-action may be exerted against neighboring T cells thathave transiently acquired peptide in association with MHC class I molecules at the site of contact of the target cells, leading to fratricidal lysis [39••,40,41].

Some lymphotrophic viruses, such as cytomegalovirus orHTLV-1, can even naturally infect CD8+ T lymphocytes,which then could become the direct target cells of CTLs [42].If physiologically relevant, such a mechanism would certainlyparticipate in decreasing the number of effector T cells following an immune response. In contrast, in the absence ofperforin-dependent cytotoxicity, not only are CTLs not killedby this mechanism but also their activation is favored, sincethe phenomenon whereby complexes of peptide with MHCclass I are captured requires TCR signaling [40].

Trans-cytolytic action could also be exerted on antigen-presenting cells (APCs). Failure to kill APCs will favor a

Cytotoxicity in lymphocyte homeostasis de Saint Basile and Fischer 551

Figure 1

Schematic representation of the secretorycytotoxic pathway in T cells. This pathway isfunctionally defective in several inheriteddisorders that feature HPS (the disorders andthe molecules contributing to HPS are boxedin the figure), that are characterized by anunbalanced expansion and activation oflymphocytes and macrophages. The positionsof the various blocks resulting from themolecular defect in each condition areindicated by red, vertical bars. In FHL,cytotoxic granules are defective in theirfunctional perforin content in 30% of patients(FHL2). In CHS, a defect in CHS1/LYST (a lysosomal trafficking regulator) leads toabnormal protein transport and/or sorting(affecting e.g. perforin, granzymes, andCTLA-4 in T cells and class II in B cells) andto an increased size of cytotoxic granules,which are unable to release their content. In some forms of GS, a defect in Rab27a (a small-GTPase, which is a vesicular trafficregulator) affects lytic granule exocytosis.

Cytotoxicactivity

FHL2

Perforin

CHSCHS1/LYST

GS

Rab27a

ER

T cell

Current Opinion in Immunology

Perforin Granule

sustained immune response. However, several studies inthe mouse, using attenuated strains of LCMV or glycopro-tein-derived epitope peptides [25••,26••], show that thismechanism is not sufficient to account for the considerablesustained expansion of the CD8+ lymphocytes and to alesser extent of the CD4+ lymphocytes observed in perforin-deficient mice compared with wild-type miceafter viral exposure.

A possible explanation is that prolonged contact betweenT cells and other T cells or between APCs and T cellsoccurring in the absence of perforin-mediated killing mayrescue activated T cells from activated-induced cell death (AICD). This rescue may involve a mitochondrial pathway or cytokines that have been shown to improvesurvival of antigen-specific T cells that were programmedto die after a primary in vivo EBV-induced immuneresponse [31] (see also Update). A recent report, however,suggests a preferential role for perforin in the control ofCD8+ T cell expansion whereas cell death and immun-odominance hierarchies appear to be under the control ofIFN-γ levels [43•]. Thus, in addition to its role as an effec-tor molecule in resistance to infection, perforin couldfunction as a regulator for T cell activation. Whether thisaction depends only on the persistence of activation signals mediated by the APCs or from indirect functionsfor perforin on T cell activation remains a subject ofdebate [44,45] (see also Update).

Whatever the precise role(s) of perforin and cytolytic granules is (are), it is unclear why mutants in the cytolyticpathway do not result in autoimmune manifestations. Onecan speculate that the way the immune response (toexogenous antigen) is triggered could play a role [33]. Thetriggering microorganisms are all intracellular, probablyinducing a specific set of innate immune responses. Inaddition, as HPS occurs in a setting of persisting infections,one can propose that selective effector T cells with highaffinity for antigen (thus excluding autoimmune clones)are primarily expanding [30••].

Are there other molecular defects underlyinghemophagocytic syndrome?Considering the so-far identified molecular defects leadingto an impairment of the secretory cytotoxic pathway, it is tempting to speculate that this pathway will also be impaired in other cases of yet unexplained HPS.Granzymes A and B and their receptors, such as the recent-ly identified mannose-6-phosphate receptor [46••], couldaccount for FHL in patients who are not perforin-defi-cient. Indeed, in approximately 50%−70% of patients withan FHL diagnosis, perforin is normally expressed and nomutation in the perforin gene can be detected, whereasNK activity is usually defective. Membrane proteinsexpressed by NK cells and CTLs with activating orinhibitory functions on T/NK cell activation, such as molecules from the KIRs/LIR-1 families, could also beconsidered [47••,48].

In XLP, the occurrence of HPS is usually triggered by anEBV infection. This disorder results from mutations in thesmall SH2-domain-containing protein, SAP/SH2D1A/DSHP, which normally associates with several cell surfacereceptors of the CD2 family, including 2B4 and SLAM(reviewed in [49••]). SLAM is expressed on T and B cells;it mediates homotypic binding and promotes T cell co-stimulation, proliferation and production of Th1cytokines. 2B4 is expressed on NK cells and CTLs; itbinds CD48, a cell surface molecule that is upregulated onEBV-infected B cells [49••]. During an EBV infection,SLAM−SLAM interactions at the interface between EBV-infected B cells and T cells may promote the developmentof EBV-specific T helper response, whereas engagementof 2B4 with CD48 will enhance lysis of infected targetcells. Impairment of the secretory cytotoxic pathway triggered through the cell surface receptor 2B4 was in factdemonstrated in these patients, as a consequence of SAPmutation [50•−52•]. Thus, this finding strengthens thefundamental role of this cytotoxic pathway in the control ofT/B cell homeostasis during viral infection.

Similarly, acquired forms of HPS can be observed duringthe course of viral infection or autoimmune diseases. Thepossibility that viral proteins may transiently interfere withthe cytotoxic pathway to limit its effect, as observed for theintracellular transport of proteins [53,54•,55], is an attractivehypothesis. In addition, in immunocompromised subjects,virus antigen cannot be properly eliminated during the firstphase of the infection, leading to the development of a verystrong ongoing immune system activation, causing inflam-matory immunopathology. HPS has been also described inassociation with malignancies, in particular lymphomas. Itis an attractive speculation to consider somatic mutationsaffecting the secretory cytotoxic pathway as causing HPSonce the cancer cell bulk has expanded.

ConclusionsSecretory and non-secretory cytotoxic functions of lymphocytes are key mechanisms in the regulation of theimmune response. Loss of their functions has profoundeffects on immune homeostasis, both in murine modelsand in patients with HPS. Fine analysis of these mutantshas provided important information on the respective roleof each pathway, their various components and the environmental context that triggers their activation.Elucidating such pathways appears critical for the under-standing of the fine regulation of T cell activation, an areathat is likely to be of therapeutic utility.

UpdateSince the submission of this review, several papers of inter-est have been published [56••,57••,58,59]. Recent workfrom Schoenberger and co-workers [56••] as well as Kaechand Ahmed [57••] have shown that a brief and single interaction between naive CTLs and stimulatory APCs issufficient to cause CTL differentiation into effectors andsubsequently into memory cells. Vivier and co-workers

552 Immunogenetics

[58] also recently provided evidence for a role for MHCclass I dependent NKR engagement in the survival of asubset of memory CD8+ T cells. Finally, Ludewig et al.[59] provide evidence suggesting that effector mechanismsother than perforin- and Fas-mediated cytotoxicity contribute to the control of dendritic cell persistence in vivo.

AcknowledgementsThis work was supported by grants from INSERM, L’Association Vaincreles Maladies Lysosomales (VML) and L’Association de Recherche Contre leCancer (ARC).

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45. Moretta L, Moretta A, Hengartner H, Zinkernagel RM: On thepathogenesis of perforin defects and related immunodeficiencies.Immunol Today 2000, 21:593-594.

46. Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, •• Barry M, Shostak I, Sawchuk T, Holmes CF et al.: Mannose

6-phosphate/insulin-like growth factor II receptor is a death

receptor for granzyme B during cytotoxic T-cell-inducedapoptosis. Cell 2000, 103:491-500.

The identification of the mannose-6-phosphate receptor as the receptor forgranzyme B, necessary to internalize granzyme B and mediate apoptosis ofthe target cells in vitro as well as allogeneic cells rejection in vivo.

47. Dietrich J, Cella M, Colonna M: Ig-like transcript 2 (ILT2)/leukocyte •• Ig-like receptor 1 (LIR1) inhibits TCR signaling and actin

cytoskeleton reorganization. J Immunol 2001, 166:2514-2521.Description of the molecular mechanisms along the signaling pathway thatfollows ILT2 ligation and leads to inhibition of T cell activation and reorgani-zation of the actin cytoskeleton.

48. Ravetch JV, Lanier LL: Immune inhibitory receptors. Science 2000,290:84-89.

49. Morra M, Howie D, Grande MS, Sayos J, Wang N, Wu C, Engel P, •• Terhorst C: X-linked lymphoproliferative disease: a progressive

immunodeficiency. Annu Rev Immunol 2001, 19:657-682.A review on the recent advances in the molecular characterization of the SAPprotein and its association with other proteins, which is altered in XLP. Thissyndrome is characterized by the occurrence of an HPS that is triggered byEBV infection. Also reported is the first description of the consequence ofvirus infection in a mouse strain in which the SAP gene has been disrupted.

50. Parolini S, Bottino C, Falco M, Augugliaro R, Giliani S, Franceschini R, • Ochs HD, Wolf H, Bonnefoy JY, Biassoni R et al.: X-linked

lymphoproliferative disease. 2B4 molecules displaying inhibitoryrather than activating function are responsible for the inability ofnatural killer cells to kill Epstein-Barr virus-infected cells. J ExpMed 2000, 192:337-346.

This paper, along with [51• ,52•], is a description of a cytotoxic defect in XLPpatients’ NK cells when triggered through the surface molecule, 2B4.

51. Nakajima H, Cella M, Bouchon A, Grierson HL, Lewis J, Duckett CS, • Cohen JI, Colonna M: Patients with X-linked lymphoproliferative

disease have a defect in 2B4 receptor-mediated NK-cellcytotoxicity. Eur J Immunol 2000, 30:3309-3318.

See annotation to [50•].

52. Tangye SG, Phillips JH, Lanier LL, Nichols KE: Functional • requirement for SAP in 2B4-mediated activation of human natural

killer cells as revealed by the X-linked lymphoproliferativesyndrome. J Immunol 2000, 165:2932-2936.

See annotation to [50•].

53. Lee BS, Alvarez X, Ishido S, Lackner AA, Jung JU: Inhibition ofintracellular transport of B-cell antigen receptor complexes byKaposi’s sarcoma-associated herpesvirus K1. J Exp Med 2000,192:11-21.

54. Ishido S, Choi JK, Lee BS, Wang C, DeMaria M, Johnson RP, • Cohen GB, Jung JU: Inhibition of natural killer cell-mediated

cytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5protein. Immunity 2000, 13:365-374.

Description of a novel viral immune evasion strategy whereby Kaposi’s-sarcoma-associated herpesvirus K5 protein achieves immune avoidance bydownregulation of cellular ligands for NK-cell-mediated cytotoxicity receptors.

55. Xu A, Bellamy AR, Taylor JA: Immobilization of the early secretorypathway by a virus glycoprotein that binds to microtubules.EMBO J 2000, 19:6465-6474.

56. van Stipdonk MJ, Lemmens EE, Schoenberger SP: Naive CTLs•• require a single brief period of antigenic stimulation for clonal

expansion and differentiation. Nat Immunol 2001, 2:423-429.This study demonstrates that a two-hour exposure to APCs either in vitro orin vivo is sufficient for priming and clonal expansion of naive CTLs into effector cells. Further divisions and differentiation of CTLs can occur without additional antigenic stimulation.

57. Kaech SM, Ahmed R: Memory CD8+ T cell differentiation: initial•• antigen encounter triggers a developmental program in naive

cells. Nat Immunol 2001, 2:415-422.This study, along with [56•• ], indicates that initial antigen contact with naiveCD8+ T cells triggers a cellular differentiation program that manifests in theformation of functional effectors and ultimately memory CD8+ T cells.

58. Ugolini S, Arpin C, Anfossi N, Walzer T, Cambiaggi A, Forster R,Lipp M, Toes RE, Melief CJ, Marvel J, Vivier E: Involvement ofinhibitory NKRs in the survival of a subset of memory-phenotypeCD8+ T cells. Nat Immunol 2001, 2:430-435.

59. Ludewig B, Bonilla WV, Dumrese T, Odermatt B, Zinkernagel RM,Hengartner H: Perforin-independent regulation of dendritic cellhomeostasis by CD8(+) T cells in vivo: implications for adaptiveimmunotherapy. Eur J Immunol 2001, 31:1772-1779.

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