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Human cytolytic T lymphocytes expressing HLA class-I-specific inhibitory receptors
Human cytolytic T lymphocytes expressing HLA class-I-specific inhibitory receptors Maria Cristina Mingari1,2, Gabriella Pietra3 and Lorenzo Moretta3,4,5 MHC class-1-specific inhibitory receptors were originally described in NK cells, in which they represent an important failsafe mechanism that induces NK cell tolerance to normal self cells. These inhibitory NK receptors (iNKRs) were subsequently found expressed on different T cell subsets, primarily CD8+ cytolytic T lymphocytes (CTLs), in which they can inhibit T cell receptor mediated functions. Some iNKR+ CTLs are HLA-Erestricted, represent oligo- or monoclonal expansions, and can play a defensive role in viral infections. Although T cell activation, in the presence of certain cytokines, can induce the expression of the CD94–NKG2A heterodimeric receptor, the mechanism leading to the expression of killer immunoglobulin-like receptors (KIRs) is still unknown. The expression of iNKRs in T cells might contribute to the prevention of apoptotic cell death, thus allowing their survival and clonal expansion in vivo. In addition, iNKR+ T cells might contribute to peripheral self-tolerance. Addresses 1 Istituto Nazionale per la Ricerca sul Cancro, L.go Rosanna Benzi, 10, 16132 Genova, Italy 2 Dipartimanento di Oncologia, Biologia e Genetica, Universita` degli Studi di Genova, L.go Rosanna Benzi, 10, 16132 Genova, Italy 3 Istituto Giannina Gaslini, L.go G. Gaslini n8 5, 16147 Genova-Quarto, Italy 4 Dipartimento di Medicina Sperimentale, Universita` degli Studi di Genova, Via L.B. Alberti n8 2, 16132 Genova, Italy 5 Centro di Eccellenza per le Ricerche Biomediche, Universita` degli Studi di Genova, V.le Benedetto XV n8 7, 16132 Genova, Italy Corresponding authors: Mingari, Maria Cristina ([email protected]); Pietra, Gabriella ([email protected])
Current Opinion in Immunology 2005, 17:312–319
molecules. Recently, a more complex scenario has emerged; the expression (or lack thereof) of activating receptors on NK cells or their ligands on target cells can also control NK cell activation and function [7,8]. The inhibitory receptors in humans include killer immunoglobulin-like receptors (KIRs), leukocyte Ig-like receptors (LIRs) and the CD94–NKG2A heterodimer, which is also present in mice. These receptors have in common the presence of intracytoplasmic immunoreceptor tyrosinebased inhibition motifs (ITIMs) that are required for their inhibitory function [9–11]. Various cell surface markers and receptors that were initially described in NK cells have also been found in different T cell subsets. In addition, expression of iNKRs can interfere with T cell activation and function or regulate T cell survival upon interaction with their HLA class I ligands. Although both CD4+ and CD8+ T cell receptor (TCR) ab+ T cells can express KIRs, KIR+ CD8+ T cells are more prominent [12–15,16,17]. NK receptors are also frequently expressed by several ‘nonconventional’ T cell populations, including TCRgd+ T cells [18], intraepithelial T lymphocytes (T-IELs), and the CD1d-restricted CD4– CD8– or CD4+ CD8– T cells, referred to as NKT cells. In this review we summarize our knowledge of the expression and function of iNKRs on human ‘conventional’ CD8+ cytolytic T lymphocytes (CTLs).
HLA class-I-specific receptors expressed by CTLs KIRs
This review comes from a themed issue on Lymphocyte effector functions Edited by Lorenzo Moretta Available online 11th April 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.03.006
Introduction MHC class-I-specific inhibitory receptors were first identified in the early 1990s in NK cells both in humans [1–4] and in mice [5,6]. iNKRs (inhibitory NK receptors) expressed on NK cells play a major role in enabling the NK cell to discriminate between normal cells (expressing adequate amounts of MHC class I molecules) and cells that have either lost or underexpress MHC class I Current Opinion in Immunology 2005, 17:312–319
The human KIR gene family, which belongs to the immunoglobulin superfamily, contains 15 genes and 2 pseudogenes that are clustered in the leukocyte receptor complex (LRC) on chromosome 19q13.4 [19]. KIRs are type I transmembrane glycoproteins and consist of either two or three immunoglobulin-like domains (designated KIR2D and KIR3D, respectively) in their extracellular region, a transmembrane portion and an intracytoplasmic tail. Notably, the cytoplasmic tail is short (S) in the activating forms, whereas it is long (L) and contains ITIMs in the inhibitory forms. Inhibitory KIRs recognize polymorphic determinants of the classical human leukocyte antigen (HLA)-A, HLA-B and HLA-C molecules. Members of the KIR2DL subfamily recognize polymorphic determinants in HLA-C molecules (i.e. Lys80- or Asn80-containing HLA-C alleles) [20], whereas KIR3DL1 interacts with HLA-BBw4 and KIR3DL2 interacts with HLA-A3 and HLA-A11 alleles (Table 1; [21,22]). www.sciencedirect.com
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Table 1 MHC class I specific inhibitory receptors expressed by human CD8+ cytolytic T lymphocytes. iNKR
Alternative denomination
CD
Chromosomal localization
Molecular family
Ligand
KIR2DL1 KIR2DL2/3 KIR3DL1 KIR3DL2 LIR1 CD94–NKG2A
p58.1 p58.2/3 p70 p140 ILT2 —
CD158a CD158b CD158e1 CD158k CD85j CD94/CD159a
19q13.4 19q13.4 19q13.4 19q13.4 19q13.4 12p12–p13
IgSF IgSF IgSF IgSF IgSF C-type lectin
HLA-CLys80 HLA-CAsn80 HLA-BBw4 HLA-A3, -A11, others? HLA-A,-B, -C, -E, -F and –G; CMV UL18 HLA-E
KIR interaction with specific HLA class I ligands expressed by potential target cells results in the suppression of cytotoxicity and cytokine secretion by KIR-bearing T cells. The molecular mechanisms involve tyrosine phosphorylation of intracytoplasmic ITIMs, which is mediated by the Src family of tyrosine kinases. This leads to recruitment and activation of the Src homology 2 domain-containing phosphatase 1 (SHP-1) at the phosphorylated ITIM. In turn, activated SHP-1 dephophorylates multiple phosphorylated intracellular proteins that are involved in the early stages of activating signalling pathways induced by TCR ligation (Figure 1). LIR1
The human LIR (also called ILT-2) family of genes is located on chromosome 19q13.4 within the leukocyte receptor complex (LCR). LIR1 is a transmembrane molecule with four immunoglobulin-like domains in the extracellular region and four ITIMs in the cytoplasmic domain. LIR1 is expressed not only by NK and T cells but also by myeloid cells [23]. It displays a ‘promiscuous’ recognition of many classical and non-classical MHC class I molecules as well as of the cytomegalovirus (CMV)-derived class I-like molecule UL18 (Table 1; [24,25]). CD94–NKG2A heterodimers
The CD94/NKG2 family of genes is located within the ‘natural killer complex’ on chromosome 12p12.3-p13.2. Unlike KIR genes, the genes encoding CD94 and NKG2 molecules display a limited polymorphism. They encode type II transmembrane proteins belonging to the C-type lectin-like family. Receptors belonging to the NKG2 family, including NKG2A, NKG2C, NKG2E and NKG2F display high homology and form disulfide-linked heterodimers with CD94. CD94–NKG2A heterodimers function as inhibitory receptors. Accordingly, NKG2A has two ITIMs in its cytoplasmic tail, which interact with SHP-1 and SHP-2 upon tyrosine phosphorylation. CD94– NKG2A is a receptor for HLA-E, the non-classical HLA class I molecule. HLA-E is assembled either with peptides derived from the processed leader sequence of classical HLA class I molecules or with some viral peptides (Table 1; [26,27]). www.sciencedirect.com
Phenotypic and functional characteristics of CD8+ TCRab+ T cells expressing iNKRs iNKR+ T cells may be induced ex novo from iNKR T cells and/or result from expansion of iNKR+ T cells consequent to in vivo T cell responses. In normal donors, KIR+CD8+ T cells are either oligoclonal or, more frequently, monoclonal T cell expansions characterized by different TCR-Vb rearrangements in different donors [28]. KIR+CD8+ T cells are present in lymph nodes, spleen and tonsils as well as in peripheral blood (PB). However, mature conventional TCRab+CD8+ T cells do not express iNKRs either in the thymus or in the umbilical cord blood [29,30]. This implies that TCR rearrangement precedes the expression of iNKRs [31,32]. In addition, the population of KIR+CD8+ T cells has been shown to increase with increasing age [30]. With respect to the surface phenotype, this population is characterized by the absence of CC chemokine receptor 7 (CCR7), CD27 and CD28 [30,33] as well as by the presence of CD57. CD45RO does not seem to be consistently expressed by KIR+CD8+ T cells, as variable proportions of these cells, isolated from different donors, can express CD45RA as well. A large fraction of KIR+CD8+ T cells are CD56+, a marker known to correlate with the acquisition of cytolytic effector function [34]. Along the same line, LIR1+ T cells do not express CCR7, thus suggesting that this cell subset is not composed of naı¨ve cells. It is conceivable that LIR1 precedes KIR expression, as the TCR repertoire of LIR1+ T cells in normal donors is not skewed towards certain TCR-Vb families, as it is for KIR+CD8+ T cells. In a recent paper, Anfossi et al. [35] provided evidence that KIR+ and LIR1+ T cells contain in their cytoplasm the cytolytic molecule perforin. Taken together, the various phenotypic data illustrated above support the notion that iNKR+ T cells are effector-memory CTLs, possibly resulting from a chronic, antigen-driven stimulation [36–39,40].
Mechanisms of cell surface expression of iNKRs on CTLs Various evidence suggests that iNKR expression on T cells occurs during or after activation of fully mature T Current Opinion in Immunology 2005, 17:312–319
314 Lymphocyte effector functions
Figure 1
Antigen recognition TCR-CD3-ζ-chain MHC class I recognition
KIR δ
P
α
ε
β
ε
ITIMs
LCK/FYN ITAMs
(d) (e)
SHP-1
P
ITIMs (b)
ZAP70
ITAMs
P P ζ
(f)
γ
Substrate
(a)
ζ
(c)
Substrate P
T-cell activation Current Opinion in Immunology
KIR-mediated inhibition of the signalling pathway initiated by the T-cell recptor. (a) Upon antigen binding to the TCR, protein tyrosine kinases of the Scr family (LCK and FYN) become activated and phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR-associated CD3-z-chain complex. (b) Phosphorylated ITAMs provide docking sites for the tyrosine kinase (SYK)-family member ZAP70 (z-chain-associated protein kinase of 70 kDa), ZAP70 becomes phosphorylated and activated by SRC kinases. (c) ZAP70 can then phosphorylate various cellular substrates; this results in T-cell activation. (d) After ligation of KIRs that recognize HLA class I molecules, immunoreceptor tyrosine-based inhibition motifs (ITIMs) become phosphorylated (this process is mediated by tyrosine kinases of the Src family). (e) Subsequently, the Src homology 2 domain-containing phosphatase 1 (SHP-1) is recruited and activated. (f) Activated SHP-1 mediates dephosphorylation of tyrosine phosphorylated substrates involved in T-cell activation.
cells. Thus, iNKR+ T cells bear surface markers that reveal previous activation. T cell clones that share identical rearrangements of TCRa and TCRb chains can stably express different KIR genes in their clonal progeny, indicating that KIR genes have been transcribed after TCR rearrangement [31,32]. Recent reports provide evidence that, at least in NK cells, the pattern of KIR expression is regulated by DNA methylation processes in CpG islands in the upstream region of KIR genes [41,42]. However, the signals determining the induction of inhibitory KIRs and LIR1 expression in T cells are still poorly understood. When a given KIR gene is expressed Current Opinion in Immunology 2005, 17:312–319
in T cells, it is stably maintained in their clonal progenies as indicated by in vitro experiments of cloning PB KIR+ T cells [13]. No convincing de novo expression of KIRs could be detected upon activation of T cells in vitro either by TCR triggering or by cytokine stimulation. The notion that repeated TCR engagement is required for stable KIR expression in vitro has not been proven. The finding that the progenies of KIR+ T cells maintain their KIR+ phenotype when cultured in IL-2, in the absence of restimulation via TCR would rather argue against this notion. In addition, data unambiguously indicating that KIR expression on CD8+ T cells is maintained in vivo www.sciencedirect.com
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through continuous encounter with antigen are still lacking [43].
Recently, Sivori et al. [44 ] reported that IL-21 induces rapid maturation of human CD34+ cell precursors towards NK cells. More importantly, IL-21 promotes acquisition of surface KIRs. However, no evidence has been obtained so far that IL-21 can also promote KIR expression in T cells. Although the signals that control KIR expression in T cells are still unknown, recent work has provided new insights into the mechanisms of control of CD94–NKG2A surface expression on T cells. First, cytokines including IL-15, TGF-b and IL-12 upregulate the expression of CD94–NKG2A heterodimers on human peripheral CD8+ T cells [45–47]. This occurs when the cytokines are added to T cells stimulated in vitro with allogeneic cells or superantigens. Jabri and collaborators [48] provided evidence that CD94–NKG2A expression is a clonal property that is developmentally acquired by T cells after both TCR expression and antigen encounter. Naturally expanded CTL clones expressing a sequence-related TCR (i.e. Vb16+ clones that, because they share the same Va12, the same CDR3 length of nine amino acids and the common four amino acid motif CVVR, are likely to display the same antigen specificity as well) have the same NKG2A expression pattern. This suggests that TCR sequences and, possibly, the antigenic specificity can determine the commitment to express NKG2A. In addition, in these T cells the expression of CD94/NKG2A could be induced by TCR engagement [48]. Along this line, one may speculate that it is possible that different KIR- T lymphocytes might be committed to KIR expression that will occur only upon delivery of suitable signals.
Control of T cell function by iNKRs Role of iNKRs in the negative regulation of T-cell activation
Various studies have indicated that the engagement of KIRs, CD94–NKG2A or LIR1 expressed on T cells can result in downregulation of TCR-mediated cell activation [49]. Thus, upon interaction with MHC class I molecules, iNKRs can inhibit T cell effector function (e.g. cytotoxicity and cytokine production). In addition, this has clearly been shown in antigen-specific HLA class I-restricted KIR+CD8+ T lymphocytes [50–53]. In a recent paper, Guerra et al. [54] investigated the effect of KIR CD158a engagement during the early steps of T cell activation. They provide evidence that KIR signalling interferes with early TCR signalling events and membrane reorganization. This prevents tyrosine phosphorylation of ZAP70 and LAT, lipid raft coalescence, TCR/CD3 accumulation at the CTL–target cell interface and actin-mediated cytoskeleton rearrangement [54]. Thus, KIR engagement results in inhibition of the membrane raft reorganization, leading to target cell protection from lysis. www.sciencedirect.com
Role of iNKRs in virus- and tumour-specific immune responses
Virus-specific CD8+ T cells can express any of a diverse collection of iNKRs. In particular, LIR1 is expressed on a large proportion of Epstein-Barr virus-, HIV-1- and CMV-specific CD8+ CTLs. Note, however, that the inhibitory capability of this receptor appears to be marginal in comparison to KIRs (see below). It has recently been shown that reactivation of human CMV increases LIR1 expression on circulating lymphocytes in lungtransplanted patients [55]. Thus, increased LIR1 expression could be a suitable marker for the early identification of CMV reactivation. In chronic infections, LIR1 signalling can partially inhibit CD8+ T cell mediated protection by suppressing IFN-g production; however, it seems to have little effect on cytolytic activity [56]. Although, LIR1+ cells outnumber the KIR+ cells within the viral-peptide-specific CD8+ T cell population [36], KIR+ T cells are also frequently detectable and have been analyzed. In this regard, our group has recently described a particular subset of CMV-specific CD8+ T cells in CMV seropositive healthy donors. This subset, restricted by the HLA-E molecule, express KIRs, CD94–NKG2A and LIR1. Cytolytic activity of these effector cells (referred to as NK-CTLs) is sharply inhibited upon KIR engagement. By contrast, CD94–NKG2A and LIR1 could not deliver efficient inhibitory signals, primarily due to their low surface expression [57,58,59,60]. As reported in various studies, tumour-specific CTLs isolated ex vivo from cancer patients can display a CD28 CD57+ iNKR+ phenotype in vivo [33,61]. In particular, in patients with melanoma or renal cell carcinoma, KIR+ CD8+ T cells specific for tumour antigens can be isolated from PB as well as from tumour sites [51–54]. Specific cytotoxicity against autologous tumour cells is strongly inhibited by KIR engagement [51]. CTLs expressing CD94–NKG2A have also been well documented in tumour patients. Thus, CD94–NKG2A+ CD8+ T cell clones have been isolated from melanoma, astrocytoma and ovarian carcinoma patients [61–63]. Although the inhibitory function of CD94–NKG2A is usually weak in T cells, IFN-g can induce upregulation of HLA-E (and HLA-G) molecules on tumour cells (e.g. ovarian carcinoma cells), thus allowing more efficient interaction between HLA-E and CD94–NKG2A, leading to inhibition of CTL function [63]. Taken together, the above data suggest that the tumourspecific CTLs present in some cancer patients might be unable to attack tumour cells because they lack the expression of inhibitory receptors. These findings also suggest that the loss of those HLA class I alleles recognized by KIRs expressed by CTLs might favour CTLmediated tumour cell lysis [51]. Current Opinion in Immunology 2005, 17:312–319
316 Lymphocyte effector functions
Role of iNKRs in the control of peripheral T cell tolerance
Several reports show that KIR-expressing CD8+ T cells specific for self antigens are present in healthy donors. This suggests that KIR has a role in the control of peripheral tolerance [33,43,64,65]. KIR expression might mediate T cell tolerance by controlling the activity of T cells that cross-react with self antigens to maintain the T cell specificities that are useful for defence mechanisms. These cells might be activated by cross-reacting foreign antigens that bind with a higher affinity to the expressed TCR. Alternatively, these CTLs might respond to self antigens when they are present at higher concentrations, which might occur as a consequence of tumour transformation or other pathological events. Accordingly, it has been proposed that KIR expression might result in an active silencing process, inducing T cell tolerance to cross-reactive self antigens accessible in the periphery. However, direct evidence that iNKRs can modulate the activation of self-reactive CTLs in vivo is still lacking. Role of iNKRs in promoting T cell survival
The apparent paradox that T cells undergo significant clonal expansion in vivo even though they express inhibitory receptors can be explained by the notion that iNKRs can protect T cells from apoptotic cell death. Thus, in addition to providing negative signals, KIRs can also favour cell growth and/or survival of KIR+ T cells [66]. Along this line, several reports indicate a possible role for KIRs in maintaining the survival of memory T cells and their resistance to activation-induced cell death (AICD). Notably, effector KIR+CD8+ T cells have higher intracytoplasmic levels of the apoptosis protective molecule Bcl-2 in comparison to KIR– LIR1+ T cells [36]. In addition, it has been shown that the interaction between ITIMs in the cytoplasmic portion of KIRs and the p85 subunit of phosphatidylinositol 3-kinase results in activation of the anti-apoptotic AKT kinase [67]. Moreover, reports indicate that KIRs can inhibit Fas-mediated apoptosis both by inducing an increase in cFLICE inhibitory protein-L (c-FLIP-L) level in CTLs and by decreasing caspase-8 activity [68]. Finally, KIRs can prevent AICD of T cells also by blocking Fas ligand induction, which follows cell stimulation. This process is associated with the blockade of protein kinase C activation involving the membrane-proximal portion of the KIR cytoplasmic tail [69].
defence mechanism against viral infections, primarily CMV infections. Early studies focused mainly on the inhibitory effect exerted by iNKRs on the effector functions of iNKR+ CTLs. More recently, the remarkable clonal expansions of HLA-E-specific NK-CTLs in vivo suggested that iNKRs could also play a role in the survival of these cells. Indeed, the iNKR-mediated anti-apoptotic effect has been well documented [66]. Other possible functions, such as the involvement of iNKRs in peripheral tolerance, remain mostly speculative and their relevance in peripheral tolerance in vivo has yet to be proven. Several additional questions still remain. For example, it is unclear what the actual stimulus (cytokines?) capable of inducing the surface expression of KIRs is, and whether this might be related to certain TCR types or particular antigen specificities. In addition, the homing capability of different iNKR+ CTLs to different tissues should be addressed in more detail. The effect of the interaction between HLA-E-specific NK-CTLs and dendritic cells (DCs) that can lead to killing of immature DCs [70] might also be relevant for the control of DC survival and function, particularly during the course of CMV infection. Answers to these core questions, together with a better definition of the functional potential of the different iNKR+ CTL subsets, will help to clarify their role in defence mechanisms and allow us to exploit their properties in immunotherapy, in view of their potential antitumour and/or anti-viral effects.
Acknowledgements This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (AIRC); Istituto Superiore di Sanita` (ISS); Ministero della Salute; Ministero dell’Istruzione dell’Universita` e della Ricerca (MIUR); and Compagnia di San Paolo. We thank Cinzia Miriello and Stefano Canu for secretarial assistance.
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Conclusions The surface expression of MHC class-I-specific inhibitory receptors, which are typical of NK cells, on T lymphocytes is an interesting and puzzling finding. Certain iNKR+ T cells that are present in some individuals, were found to be HLA-E-restricted, thus revealing a novel role for HLA-E molecules. The oligo- or monoclonality of these cells, together with their specificity for a limited number of HLA-E-binding peptides, including viral peptides, suggests that they are an additional Current Opinion in Immunology 2005, 17:312–319
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of polymorphic HLA-B moelcules. J Exp Med 1994, 180:537-544. 22. Pende D, Biassoni R, Cantoni C, Verdiani S, Falco M, Di Donato C, Accame L, Bottino C, Moretta A, Moretta L: The natural Killer cell receptor specific for HLA-A allotypes. A novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140kD disulphide-linked dimer. J Exp Med 1996, 184:505-518. 23. Colonna M, Navarro F, Bellon T, Llano M, Garcia P, Samaridis J, Angman L, Cella M, Lopez-Botet M: A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J Exp Med 1997, 186:1809-1818. 24. Cosman D, Fanger N, Borges L, Kubin M, Chin W, Peterson L, Hsu ML: A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 1997, 7:273-282. 25. Willcox BE, Thomas LM, Bjorkman PJ: Crystal structure of HLAA2 bound to LIR-1, a host and viral major histocompatibility complex receptor. Nat Immunol 2003, 4:913-919. 26. Braud VM, Allan DS, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH et al.: HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 1998, 391:795-799. 27. Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, Gadola S, Cerundolo V, Borysiewicz LK, McMichael AJ, Wilkinson GW: Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 2000, 287:1031-1033. 28. Mingari MC, Schiavetti F, Ponte M, Vitale C, Maggi E, Romagnani S, Demarest J, Pantaleo G, Fauci AS, Moretta L: Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations. Proc Natl Acad Sci USA 1996, 93:12433-12438. 29. Mingari MC, Ponte M, Cantoni C, Vitale C, Schiavetti F, Bertone S, Bellomo R, Cappai AT, Biassoni R: HLA-class I-specific inhibitory receptors in human cytolytic T lymphocytes: molecular characterization, distribution in lymphoid tissues and co-expression by individual T cells. Int Immunol 1997, 9:485-491. 30. Anfossi N, Pascal V, Vivier E, Ugolini S: Biology of T memory type 1 cells. Immunol Rev 2001, 181:269-278. 31. Vely F, Peyrat M, Couedel C, Morcet J, Halary F, Davodeau F, Romagne F, Scotet E, Saulquin X, Houssaint E et al.: Regulation of inhibitory and activating killer-cell Ig-like receptor expression occurs in T cells after termination of TCR rearrangements. J Immunol 2001, 166:2487-2494. 32. Uhrberg M, Valiante NM, Young NT, Lanier LL, Phillips JH, Parham P: The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical TCR rearrangement express highly diverse killer cell Ig-like receptor patterns. J Immunol 2001, 166:3923-3932. 33. Speiser DE, Valmori D, Rimoldi D, Pittet MJ, Lienard D, Cerundolo V, MacDonald HR, Cerottini JC, Romero P: CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. Eur J Immunol 1999, 29:1990-1999. 34. Pittet MJ, Speiser DE, Valmori D, Cerottini JC, Romero P: Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression. J Immunol 2000, 164:1148-1152. 35. Anfossi N, Doisne JM, Peyrat MA, Ugolini S, Bonnaud O, Bossy D, Pitard V, Merville P, Moreau JF, Delfraissy JF et al.: Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells. J Immunol 2004, 173:7223-7299. A recent study that analyses the expression of perforin and the functional properties of human iNKR+ CD8+ TCRab+ T cells isolated ex vivo. Current Opinion in Immunology 2005, 17:312–319
318 Lymphocyte effector functions
36. Young NT, Uhrberg M, Phillips JH, Lanier LL, Parham P: Differential expression of leukocyte receptor complexencoded Ig-like receptors correlates with the transition from effector to memory CTL. J Immunol 2001, 166:3933-3941. 37. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999, 401:708-712. 38. Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, Ogg GS, King A, Lechner F, Spina CA et al.: Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002, 8:379-385. 39. Rufer N, Zippelius A, Batard P, Pittet MJ, Kurth I, Corthesy P, Cerottini JC, Leyvraz S, Roosnek E, Nabholz M, Romero P: Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions. Blood 2003, 102:1779-1787. 40. Sallusto F, Geginat J, Lanzavecchia A: Central memory and effector memory t cell subset: function, generation and maintenance. Annu Rev Immunol 2004, 22:745-763. This review addresses the heterogeneity of TCM and TEM, their differentiation stages, and the models for their generation and maintenance in humans and mice. 41. Santourlidis S, Trompeter HI, Weinhold S, Eisermann B, Meyer KL, Wernet P, Uhrberg M: Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells. J Immunol 2002, 169:4253-4261. 42. Chan HW, Kurago ZB, Stewart CA, Wilson MJ, Martin MP, Mace BE, Carrington M, Trowsdale J, Lutz CT: DNA methylation maintains allele-specific KIR gene expression in human natural killer cells. J Exp Med 2003, 197:245-255. This interesting study, together with previous one (reference [41]) illustrates the role of DNA methylation in determining KIR gene expression. The authors also provide evidence that this mechanism is responsible for maintaining the monoallelic expression of KIR. 43. Huard B, Karlsson L: KIR expression on self-reactive CD8+ T cells is controlled by T-cell receptor engagement. Nature 2000, 403:325-328. 44. Sivori S, Cantoni C, Parolini S, Marcenaro E, Conte R, Moretta L, Moretta A: IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors. Eur J Immunol 2003, 33:3439-3447. This study shows that culture of CD34+ precursors in the presence of IL21 results in accelerated cell maturation towards NK cells. It also provides the first evidence that KIRs can be expressed ‘de novo’ in vitro. 45. Mingari MC, Ponte M, Bertone S, Schiavetti F, Vitale C, Bellomo R, Moretta A, Moretta L: HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin-15 induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells. Proc Natl Acad Sci USA 1998, 95:1172-1177. 46. Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari MC: Transforming growth factor-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol 1999, 29:23-29. 47. Derre L, Corvaisier M, Pandolfino MC, Diez E, Jotereau F, Gervois N: Expression of CD94/NKG2-A on human T lymphocytes is induced by IL-12: implications for adoptive immunotherapy. J Immunol 2002, 168:4864-4870. 48. Jabri B, Selby JM, Negulescu H, Lee L, Roberts AI, Beavis A, Lopez-Botet M, Ebert EC, Winchester RJ: TCR specificity dictates CD94/NKG2A expression by human CTL. Immunity 2002, 17:487-499. 49. D’Andrea A, Chang C, Phillips JH, Lanier LL: Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J Exp Med 1996, 184:789-794. 50. De Maria A, Ferraris A, Guastella M, Pilia S, Cantoni C, Polero L, Mingari MC, Bassetti D, Fauci AS, Moretta L: Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: impairment of Current Opinion in Immunology 2005, 17:312–319
specific cytolytic functions. Proc Natl Acad Sci USA 1997, 94:10285-10288. 51. Ikeda H, Lethe B, Lehmann F, van Baren N, Baurain JF, de Smet C, Chambost H, Vitale M, Moretta A, Boon T, Coulie PG: Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity 1997, 6:199-208. 52. Bakker AB, Phillips JH, Figdor CG, Lanier LL: Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, T cells, and antigenspecific CTL. J Immunol 1998, 160:5239-5245 (1998). 53. Guerra N, Guillard M, Angevin E, Echchakir H, Escudier B, Moretta A, Chouaib S, Caignard A: Killer inhibitory receptor (CD158b) modulates the lytic activity of tumor-specific T lymphocytes infiltrating renal cell carcinomas. Blood 2000, 95:2883-2889. 54. Guerra N, Michel F, Gati A, Gaudin C, Mishal Z, Escudier B, Acuto O, Chouaib S, Caignard A: Engagement of the inhibitory receptor CD158a interrupts TCR signaling, preventing dynamic membrane reorganization in CTL/tumor cell interaction. Blood 2002, 100:2874-2881. 55. Berg L, Riise GC, Cosman D, Bergstrom T, Olofsson S, Karre K, Carbone E: LIR-1 expression on lymphocytes, and cytomegalovirus disease in lung-transplant recipients. Lancet 2003, 361:1099-1101. 56. Ince N, Harnisch B, Xu Z, Lange C, Moretta L, Lederman M, Lieberman J: Increased expression of the natural killer cell inhibitory receptor CD85j/ILT2 on antigen specific effector CD8 T cells and its impact on CD8 T cell function. Immunology 2004, 112:531-542. This study shows that LIR1 engagement can inhibit IFN-g production but not cytolytic activity of virus-specific CTLs. 57. Pietra G, Romagnani C, Falco M, Vitale M, Castriconi R, Pende D, Millo E, Anfossi S, Biassoni R, Moretta L, Mingari MC: The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR a/b-mediated recognition. Eur J Immunol 2001, 31:3687-3693. 58. Romagnani C, Pietra G, Falco M, Millo E, Mazzarino P, Biassoni R, Moretta A, Moretta L, Mingari MC: Identification of HLA-Especific alloreactive T lymphocytes: a cell subset that undergoes preferential expansion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells. Proc Natl Acad Sci USA 2002, 99:11328-11333. 59. Moretta L, Romagnani C, Pietra G, Moretta A, Mingari MC: NK-CTLs, a novel HLA-E-restricted T-cell subset. Trends Immunol 2003, 24:136-143. A review describing the recently identified, HLA-specific CTL subset. Their anti-viral and anti-tumour activity are described as well as their possible negative effect in transplant rejection. 60. Pietra G, Romagnani C, Mazzarino P, Falco M, Millo E, Moretta A, Moretta L, Mingari MC: HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes. Proc Natl Acad Sci USA 2003, 100:10896-10901. This article characterizes the peptide specificity of NK-CTLs, a novel HLA-E restricted iNKR+ T cell subset, suggesting that they may play a role in defences against CMV and, possibly, other pathogens. 61. Speiser DE, Pittet MJ, Valmori D, Dunbar R, Rimoldi D, Lienard D, MacDonald HR, Cerottini JC, Cerundolo V, Romero P: In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J Exp Med 1999, 190:775-782. 62. Perrin G, Speiser D, Porret A, Quiquerez AL, Walker PR, Dietrich PY: Sister cytotoxic CD8+ T cell clones differing in natural killer inhibitory receptor expression in human astrocytoma. Immunol Lett 2002, 81:125-132. 63. Malmberg KJ, Levitsky V, Norell H, de Matos CT, Carlsten M, Schedvins K, Rabbani H, Moretta A, Soderstrom K, Levitskaya J, Kiessling R: IFN-gamma protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2Adependent mechanism. J Clin Invest 2002, 110:1515-1523. 64. Halary F, Peyrat MA, Champagne E, Lopez-Botet M, Moretta A, Moretta L, Vie H, Fournie JJ, Bonneville M: Control of selfwww.sciencedirect.com
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reactive cytotoxic T lymphocytes expressing gamma delta T cell receptors by natural killer inhibitory receptors. Eur J Immunol 1997, 27:2812-2821.
cytotoxic T-lymphocyte susceptibility to tumor-mediated activation-induced cell death by interfering with Fas signaling. Cancer Res 2003, 63:7475-7482.
65. Huard B, Karlsson L: A subpopulation of CD8+ T cells specific for melanocyte differentiation antigens expresses killer inhibitory receptors (KIR) in healthy donors: evidence for a role of KIR in the control of peripheral tolerance. Eur J Immunol 2000, 30:1665-1675.
69. Chwae YJ, Chang MJ, Park SM, Yoon H, Park HJ, Kim SJ, Kim J: Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells. J Immunol 2002, 169:3726-3735.
66. Ugolini S, Arpin C, Anfossi N, Walzer T, Cambiaggi A, Forster R, Lipp M, Toes RE, Melief CJ, Marvel J, Vivier E: Involvement of inhibitory NKRs in the survival of a subset of memoryphenotype CD8+ T cells. Nat Immunol 2001, 2:430-435.
70. Pietra G, Romagnani C, Mazzarino P, Millo E, Moretta L, Mingari MC: Comparative analysis of the NK or NK-CTL-mediated lysis of immature or mature autologous dendritic cells. Eur J Immunol 2003, 33:3427-3432. Different from NK cells, NK-CTLs do not kill autologous DCs presenting HLA-E-bound self peptides. However, when DCs carry HLA-E-bound foreign peptides, immature DCs are efficiently killed. This might occur in CMV infection and may result in a NK-CTL mediated control of DC survival and function. Indeed, only those DCs expressing adequate amounts of HLA-class I (i.e. mature DCs) will be resistant to NK-CTLmediated lysis thanks to the engagement of iNKRs.
67. Marti F, Xu CW, Selvakumar A, Brent R, Dupont B, King PD: LCKphosphorylated human killer cell-inhibitory receptors recruit and activate phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 1998, 95:11810-11815. 68. Gati A, Guerra N, Gaudin C, Da Rocha S, Escudier B, Lecluse Y, Bettaieb A, Chouaib S, Caignard A: CD158 receptor controls
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Current Opinion in Immunology 2005, 17:312–319
Current Opinion in Immunology 2005, 17:312–319
molecules. Recently, a more complex scenario has emerged; the expression (or lack thereof) of activating receptors on NK cells or their ligands on target cells can also control NK cell activation and function [7,8]. The inhibitory receptors in humans include killer immunoglobulin-like receptors (KIRs), leukocyte Ig-like receptors (LIRs) and the CD94–NKG2A heterodimer, which is also present in mice. These receptors have in common the presence of intracytoplasmic immunoreceptor tyrosinebased inhibition motifs (ITIMs) that are required for their inhibitory function [9–11]. Various cell surface markers and receptors that were initially described in NK cells have also been found in different T cell subsets. In addition, expression of iNKRs can interfere with T cell activation and function or regulate T cell survival upon interaction with their HLA class I ligands. Although both CD4+ and CD8+ T cell receptor (TCR) ab+ T cells can express KIRs, KIR+ CD8+ T cells are more prominent [12–15,16,17]. NK receptors are also frequently expressed by several ‘nonconventional’ T cell populations, including TCRgd+ T cells [18], intraepithelial T lymphocytes (T-IELs), and the CD1d-restricted CD4– CD8– or CD4+ CD8– T cells, referred to as NKT cells. In this review we summarize our knowledge of the expression and function of iNKRs on human ‘conventional’ CD8+ cytolytic T lymphocytes (CTLs).
HLA class-I-specific receptors expressed by CTLs KIRs
This review comes from a themed issue on Lymphocyte effector functions Edited by Lorenzo Moretta Available online 11th April 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.03.006
Introduction MHC class-I-specific inhibitory receptors were first identified in the early 1990s in NK cells both in humans [1–4] and in mice [5,6]. iNKRs (inhibitory NK receptors) expressed on NK cells play a major role in enabling the NK cell to discriminate between normal cells (expressing adequate amounts of MHC class I molecules) and cells that have either lost or underexpress MHC class I Current Opinion in Immunology 2005, 17:312–319
The human KIR gene family, which belongs to the immunoglobulin superfamily, contains 15 genes and 2 pseudogenes that are clustered in the leukocyte receptor complex (LRC) on chromosome 19q13.4 [19]. KIRs are type I transmembrane glycoproteins and consist of either two or three immunoglobulin-like domains (designated KIR2D and KIR3D, respectively) in their extracellular region, a transmembrane portion and an intracytoplasmic tail. Notably, the cytoplasmic tail is short (S) in the activating forms, whereas it is long (L) and contains ITIMs in the inhibitory forms. Inhibitory KIRs recognize polymorphic determinants of the classical human leukocyte antigen (HLA)-A, HLA-B and HLA-C molecules. Members of the KIR2DL subfamily recognize polymorphic determinants in HLA-C molecules (i.e. Lys80- or Asn80-containing HLA-C alleles) [20], whereas KIR3DL1 interacts with HLA-BBw4 and KIR3DL2 interacts with HLA-A3 and HLA-A11 alleles (Table 1; [21,22]). www.sciencedirect.com
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Table 1 MHC class I specific inhibitory receptors expressed by human CD8+ cytolytic T lymphocytes. iNKR
Alternative denomination
CD
Chromosomal localization
Molecular family
Ligand
KIR2DL1 KIR2DL2/3 KIR3DL1 KIR3DL2 LIR1 CD94–NKG2A
p58.1 p58.2/3 p70 p140 ILT2 —
CD158a CD158b CD158e1 CD158k CD85j CD94/CD159a
19q13.4 19q13.4 19q13.4 19q13.4 19q13.4 12p12–p13
IgSF IgSF IgSF IgSF IgSF C-type lectin
HLA-CLys80 HLA-CAsn80 HLA-BBw4 HLA-A3, -A11, others? HLA-A,-B, -C, -E, -F and –G; CMV UL18 HLA-E
KIR interaction with specific HLA class I ligands expressed by potential target cells results in the suppression of cytotoxicity and cytokine secretion by KIR-bearing T cells. The molecular mechanisms involve tyrosine phosphorylation of intracytoplasmic ITIMs, which is mediated by the Src family of tyrosine kinases. This leads to recruitment and activation of the Src homology 2 domain-containing phosphatase 1 (SHP-1) at the phosphorylated ITIM. In turn, activated SHP-1 dephophorylates multiple phosphorylated intracellular proteins that are involved in the early stages of activating signalling pathways induced by TCR ligation (Figure 1). LIR1
The human LIR (also called ILT-2) family of genes is located on chromosome 19q13.4 within the leukocyte receptor complex (LCR). LIR1 is a transmembrane molecule with four immunoglobulin-like domains in the extracellular region and four ITIMs in the cytoplasmic domain. LIR1 is expressed not only by NK and T cells but also by myeloid cells [23]. It displays a ‘promiscuous’ recognition of many classical and non-classical MHC class I molecules as well as of the cytomegalovirus (CMV)-derived class I-like molecule UL18 (Table 1; [24,25]). CD94–NKG2A heterodimers
The CD94/NKG2 family of genes is located within the ‘natural killer complex’ on chromosome 12p12.3-p13.2. Unlike KIR genes, the genes encoding CD94 and NKG2 molecules display a limited polymorphism. They encode type II transmembrane proteins belonging to the C-type lectin-like family. Receptors belonging to the NKG2 family, including NKG2A, NKG2C, NKG2E and NKG2F display high homology and form disulfide-linked heterodimers with CD94. CD94–NKG2A heterodimers function as inhibitory receptors. Accordingly, NKG2A has two ITIMs in its cytoplasmic tail, which interact with SHP-1 and SHP-2 upon tyrosine phosphorylation. CD94– NKG2A is a receptor for HLA-E, the non-classical HLA class I molecule. HLA-E is assembled either with peptides derived from the processed leader sequence of classical HLA class I molecules or with some viral peptides (Table 1; [26,27]). www.sciencedirect.com
Phenotypic and functional characteristics of CD8+ TCRab+ T cells expressing iNKRs iNKR+ T cells may be induced ex novo from iNKR T cells and/or result from expansion of iNKR+ T cells consequent to in vivo T cell responses. In normal donors, KIR+CD8+ T cells are either oligoclonal or, more frequently, monoclonal T cell expansions characterized by different TCR-Vb rearrangements in different donors [28]. KIR+CD8+ T cells are present in lymph nodes, spleen and tonsils as well as in peripheral blood (PB). However, mature conventional TCRab+CD8+ T cells do not express iNKRs either in the thymus or in the umbilical cord blood [29,30]. This implies that TCR rearrangement precedes the expression of iNKRs [31,32]. In addition, the population of KIR+CD8+ T cells has been shown to increase with increasing age [30]. With respect to the surface phenotype, this population is characterized by the absence of CC chemokine receptor 7 (CCR7), CD27 and CD28 [30,33] as well as by the presence of CD57. CD45RO does not seem to be consistently expressed by KIR+CD8+ T cells, as variable proportions of these cells, isolated from different donors, can express CD45RA as well. A large fraction of KIR+CD8+ T cells are CD56+, a marker known to correlate with the acquisition of cytolytic effector function [34]. Along the same line, LIR1+ T cells do not express CCR7, thus suggesting that this cell subset is not composed of naı¨ve cells. It is conceivable that LIR1 precedes KIR expression, as the TCR repertoire of LIR1+ T cells in normal donors is not skewed towards certain TCR-Vb families, as it is for KIR+CD8+ T cells. In a recent paper, Anfossi et al. [35] provided evidence that KIR+ and LIR1+ T cells contain in their cytoplasm the cytolytic molecule perforin. Taken together, the various phenotypic data illustrated above support the notion that iNKR+ T cells are effector-memory CTLs, possibly resulting from a chronic, antigen-driven stimulation [36–39,40].
Mechanisms of cell surface expression of iNKRs on CTLs Various evidence suggests that iNKR expression on T cells occurs during or after activation of fully mature T Current Opinion in Immunology 2005, 17:312–319
314 Lymphocyte effector functions
Figure 1
Antigen recognition TCR-CD3-ζ-chain MHC class I recognition
KIR δ
P
α
ε
β
ε
ITIMs
LCK/FYN ITAMs
(d) (e)
SHP-1
P
ITIMs (b)
ZAP70
ITAMs
P P ζ
(f)
γ
Substrate
(a)
ζ
(c)
Substrate P
T-cell activation Current Opinion in Immunology
KIR-mediated inhibition of the signalling pathway initiated by the T-cell recptor. (a) Upon antigen binding to the TCR, protein tyrosine kinases of the Scr family (LCK and FYN) become activated and phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR-associated CD3-z-chain complex. (b) Phosphorylated ITAMs provide docking sites for the tyrosine kinase (SYK)-family member ZAP70 (z-chain-associated protein kinase of 70 kDa), ZAP70 becomes phosphorylated and activated by SRC kinases. (c) ZAP70 can then phosphorylate various cellular substrates; this results in T-cell activation. (d) After ligation of KIRs that recognize HLA class I molecules, immunoreceptor tyrosine-based inhibition motifs (ITIMs) become phosphorylated (this process is mediated by tyrosine kinases of the Src family). (e) Subsequently, the Src homology 2 domain-containing phosphatase 1 (SHP-1) is recruited and activated. (f) Activated SHP-1 mediates dephosphorylation of tyrosine phosphorylated substrates involved in T-cell activation.
cells. Thus, iNKR+ T cells bear surface markers that reveal previous activation. T cell clones that share identical rearrangements of TCRa and TCRb chains can stably express different KIR genes in their clonal progeny, indicating that KIR genes have been transcribed after TCR rearrangement [31,32]. Recent reports provide evidence that, at least in NK cells, the pattern of KIR expression is regulated by DNA methylation processes in CpG islands in the upstream region of KIR genes [41,42]. However, the signals determining the induction of inhibitory KIRs and LIR1 expression in T cells are still poorly understood. When a given KIR gene is expressed Current Opinion in Immunology 2005, 17:312–319
in T cells, it is stably maintained in their clonal progenies as indicated by in vitro experiments of cloning PB KIR+ T cells [13]. No convincing de novo expression of KIRs could be detected upon activation of T cells in vitro either by TCR triggering or by cytokine stimulation. The notion that repeated TCR engagement is required for stable KIR expression in vitro has not been proven. The finding that the progenies of KIR+ T cells maintain their KIR+ phenotype when cultured in IL-2, in the absence of restimulation via TCR would rather argue against this notion. In addition, data unambiguously indicating that KIR expression on CD8+ T cells is maintained in vivo www.sciencedirect.com
Human CTLs expressing iNKRs Mingari, Pietra and Moretta 315
through continuous encounter with antigen are still lacking [43].
Recently, Sivori et al. [44 ] reported that IL-21 induces rapid maturation of human CD34+ cell precursors towards NK cells. More importantly, IL-21 promotes acquisition of surface KIRs. However, no evidence has been obtained so far that IL-21 can also promote KIR expression in T cells. Although the signals that control KIR expression in T cells are still unknown, recent work has provided new insights into the mechanisms of control of CD94–NKG2A surface expression on T cells. First, cytokines including IL-15, TGF-b and IL-12 upregulate the expression of CD94–NKG2A heterodimers on human peripheral CD8+ T cells [45–47]. This occurs when the cytokines are added to T cells stimulated in vitro with allogeneic cells or superantigens. Jabri and collaborators [48] provided evidence that CD94–NKG2A expression is a clonal property that is developmentally acquired by T cells after both TCR expression and antigen encounter. Naturally expanded CTL clones expressing a sequence-related TCR (i.e. Vb16+ clones that, because they share the same Va12, the same CDR3 length of nine amino acids and the common four amino acid motif CVVR, are likely to display the same antigen specificity as well) have the same NKG2A expression pattern. This suggests that TCR sequences and, possibly, the antigenic specificity can determine the commitment to express NKG2A. In addition, in these T cells the expression of CD94/NKG2A could be induced by TCR engagement [48]. Along this line, one may speculate that it is possible that different KIR- T lymphocytes might be committed to KIR expression that will occur only upon delivery of suitable signals.
Control of T cell function by iNKRs Role of iNKRs in the negative regulation of T-cell activation
Various studies have indicated that the engagement of KIRs, CD94–NKG2A or LIR1 expressed on T cells can result in downregulation of TCR-mediated cell activation [49]. Thus, upon interaction with MHC class I molecules, iNKRs can inhibit T cell effector function (e.g. cytotoxicity and cytokine production). In addition, this has clearly been shown in antigen-specific HLA class I-restricted KIR+CD8+ T lymphocytes [50–53]. In a recent paper, Guerra et al. [54] investigated the effect of KIR CD158a engagement during the early steps of T cell activation. They provide evidence that KIR signalling interferes with early TCR signalling events and membrane reorganization. This prevents tyrosine phosphorylation of ZAP70 and LAT, lipid raft coalescence, TCR/CD3 accumulation at the CTL–target cell interface and actin-mediated cytoskeleton rearrangement [54]. Thus, KIR engagement results in inhibition of the membrane raft reorganization, leading to target cell protection from lysis. www.sciencedirect.com
Role of iNKRs in virus- and tumour-specific immune responses
Virus-specific CD8+ T cells can express any of a diverse collection of iNKRs. In particular, LIR1 is expressed on a large proportion of Epstein-Barr virus-, HIV-1- and CMV-specific CD8+ CTLs. Note, however, that the inhibitory capability of this receptor appears to be marginal in comparison to KIRs (see below). It has recently been shown that reactivation of human CMV increases LIR1 expression on circulating lymphocytes in lungtransplanted patients [55]. Thus, increased LIR1 expression could be a suitable marker for the early identification of CMV reactivation. In chronic infections, LIR1 signalling can partially inhibit CD8+ T cell mediated protection by suppressing IFN-g production; however, it seems to have little effect on cytolytic activity [56]. Although, LIR1+ cells outnumber the KIR+ cells within the viral-peptide-specific CD8+ T cell population [36], KIR+ T cells are also frequently detectable and have been analyzed. In this regard, our group has recently described a particular subset of CMV-specific CD8+ T cells in CMV seropositive healthy donors. This subset, restricted by the HLA-E molecule, express KIRs, CD94–NKG2A and LIR1. Cytolytic activity of these effector cells (referred to as NK-CTLs) is sharply inhibited upon KIR engagement. By contrast, CD94–NKG2A and LIR1 could not deliver efficient inhibitory signals, primarily due to their low surface expression [57,58,59,60]. As reported in various studies, tumour-specific CTLs isolated ex vivo from cancer patients can display a CD28 CD57+ iNKR+ phenotype in vivo [33,61]. In particular, in patients with melanoma or renal cell carcinoma, KIR+ CD8+ T cells specific for tumour antigens can be isolated from PB as well as from tumour sites [51–54]. Specific cytotoxicity against autologous tumour cells is strongly inhibited by KIR engagement [51]. CTLs expressing CD94–NKG2A have also been well documented in tumour patients. Thus, CD94–NKG2A+ CD8+ T cell clones have been isolated from melanoma, astrocytoma and ovarian carcinoma patients [61–63]. Although the inhibitory function of CD94–NKG2A is usually weak in T cells, IFN-g can induce upregulation of HLA-E (and HLA-G) molecules on tumour cells (e.g. ovarian carcinoma cells), thus allowing more efficient interaction between HLA-E and CD94–NKG2A, leading to inhibition of CTL function [63]. Taken together, the above data suggest that the tumourspecific CTLs present in some cancer patients might be unable to attack tumour cells because they lack the expression of inhibitory receptors. These findings also suggest that the loss of those HLA class I alleles recognized by KIRs expressed by CTLs might favour CTLmediated tumour cell lysis [51]. Current Opinion in Immunology 2005, 17:312–319
316 Lymphocyte effector functions
Role of iNKRs in the control of peripheral T cell tolerance
Several reports show that KIR-expressing CD8+ T cells specific for self antigens are present in healthy donors. This suggests that KIR has a role in the control of peripheral tolerance [33,43,64,65]. KIR expression might mediate T cell tolerance by controlling the activity of T cells that cross-react with self antigens to maintain the T cell specificities that are useful for defence mechanisms. These cells might be activated by cross-reacting foreign antigens that bind with a higher affinity to the expressed TCR. Alternatively, these CTLs might respond to self antigens when they are present at higher concentrations, which might occur as a consequence of tumour transformation or other pathological events. Accordingly, it has been proposed that KIR expression might result in an active silencing process, inducing T cell tolerance to cross-reactive self antigens accessible in the periphery. However, direct evidence that iNKRs can modulate the activation of self-reactive CTLs in vivo is still lacking. Role of iNKRs in promoting T cell survival
The apparent paradox that T cells undergo significant clonal expansion in vivo even though they express inhibitory receptors can be explained by the notion that iNKRs can protect T cells from apoptotic cell death. Thus, in addition to providing negative signals, KIRs can also favour cell growth and/or survival of KIR+ T cells [66]. Along this line, several reports indicate a possible role for KIRs in maintaining the survival of memory T cells and their resistance to activation-induced cell death (AICD). Notably, effector KIR+CD8+ T cells have higher intracytoplasmic levels of the apoptosis protective molecule Bcl-2 in comparison to KIR– LIR1+ T cells [36]. In addition, it has been shown that the interaction between ITIMs in the cytoplasmic portion of KIRs and the p85 subunit of phosphatidylinositol 3-kinase results in activation of the anti-apoptotic AKT kinase [67]. Moreover, reports indicate that KIRs can inhibit Fas-mediated apoptosis both by inducing an increase in cFLICE inhibitory protein-L (c-FLIP-L) level in CTLs and by decreasing caspase-8 activity [68]. Finally, KIRs can prevent AICD of T cells also by blocking Fas ligand induction, which follows cell stimulation. This process is associated with the blockade of protein kinase C activation involving the membrane-proximal portion of the KIR cytoplasmic tail [69].
defence mechanism against viral infections, primarily CMV infections. Early studies focused mainly on the inhibitory effect exerted by iNKRs on the effector functions of iNKR+ CTLs. More recently, the remarkable clonal expansions of HLA-E-specific NK-CTLs in vivo suggested that iNKRs could also play a role in the survival of these cells. Indeed, the iNKR-mediated anti-apoptotic effect has been well documented [66]. Other possible functions, such as the involvement of iNKRs in peripheral tolerance, remain mostly speculative and their relevance in peripheral tolerance in vivo has yet to be proven. Several additional questions still remain. For example, it is unclear what the actual stimulus (cytokines?) capable of inducing the surface expression of KIRs is, and whether this might be related to certain TCR types or particular antigen specificities. In addition, the homing capability of different iNKR+ CTLs to different tissues should be addressed in more detail. The effect of the interaction between HLA-E-specific NK-CTLs and dendritic cells (DCs) that can lead to killing of immature DCs [70] might also be relevant for the control of DC survival and function, particularly during the course of CMV infection. Answers to these core questions, together with a better definition of the functional potential of the different iNKR+ CTL subsets, will help to clarify their role in defence mechanisms and allow us to exploit their properties in immunotherapy, in view of their potential antitumour and/or anti-viral effects.
Acknowledgements This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (AIRC); Istituto Superiore di Sanita` (ISS); Ministero della Salute; Ministero dell’Istruzione dell’Universita` e della Ricerca (MIUR); and Compagnia di San Paolo. We thank Cinzia Miriello and Stefano Canu for secretarial assistance.
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