HLA Class I-specific Inhibitory Receptors Research Articles

Underlying mechanisms of evasion from NK cells as rationale for improvement of NK cell-based immunotherapies.

Natural killer (NK) cells belong to the family of innate immune cells with the capacity to recognize and kill tumor cells. Different phenotypes and functional properties of NK cells have been described in tumor patients, which could be shaped by the tumor microenvironment. The discovery of HLA class I-specific inhibitory receptors controlling NK cell activity paved the way to the fundamental concept of modulating immune responses that are regulated by an array of inhibitory receptors, and emphasized the importance to explore the potential of NK cells in cancer therapy. Although a whole range of NK cell-based approaches are currently being developed, there are still major challenges that need to be overcome for improved efficacy of these therapies. These include escape of tumor cells from NK cell recognition due to their expression of inhibitory molecules, immune suppressive signals of NK cells, reduced NK cell infiltration of tumors, an immune suppressive micromilieu and limited in vivo persistence of NK cells. Therefore, this review provides an overview about the NK cell biology, alterations of NK cell activities, changes in tumor cells and the tumor microenvironment contributing to immune escape or immune surveillance by NK cells and their underlying molecular mechanisms as well as the current status and novel aspects of NK cell-based therapeutic strategies including their genetic engineering and their combination with conventional treatment options to overcome tumor-mediated evasion strategies and improve therapy efficacy.

Inhibitory Receptors and Checkpoints in Human NK Cells, Implications for the Immunotherapy of Cancer.

The highly destructive mechanisms by which the immune system faces microbial infections is under the control of a series of inhibitory receptors. While most of these receptors prevent unwanted/excessive responses of individual effector cells, others play a more general role in immunity, acting as true inhibitory checkpoints controlling both innate and adaptive immunity. Regarding human NK cells, their function is finely regulated by HLA-class I-specific inhibitory receptors which allow discrimination between HLA-I+, healthy cells and tumor or virus-infected cells displaying loss or substantial alterations of HLA-I molecules, including allelic losses that are sensed by KIRs. A number of non-HLA-specific receptors have been identified which recognize cell surface or extracellular matrix ligands and may contribute to the physiologic control of immune responses and tolerance. Among these receptors, Siglec 7 (p75/AIRM-1), LAIR-1 and IRp60, recognize ligands including sialic acids, extracellular matrix/collagen or aminophospholipids, respectively. These ligands may be expressed at the surface of tumor cells, thus inhibiting NK cell function. Expression of the PD-1 checkpoint by NK cells requires particular cytokines (IL-15, IL-12, IL-18) together with cortisol, a combination that may occur in the microenvironment of different tumors. Blocking of single or combinations of inhibitory receptors unleashes NK cells and restore their anti-tumor activity, with obvious implications for tumor immunotherapy.

Exploiting Human NK Cells in Tumor Therapy.

NK cells play an important role in the innate defenses against tumor growth and metastases. Human NK cell activation and function are regulated by an array of HLA class I-specific inhibitory receptors and activating receptors recognizing ligands expressed de novo on tumor or virus-infected cells. NK cells have been exploited in immunotherapy of cancer, including: (1) the in vivo infusion of IL-2 or IL-15, cytokines inducing activation and proliferation of NK cells that are frequently impaired in cancer patients. Nonetheless, the significant toxicity experienced, primarily with IL-2, limited their use except for combination therapies, e.g., IL-15 with checkpoint inhibitors; (2) the adoptive immunotherapy with cytokine-induced NK cells had effect on some melanoma metastases (lung), while other localizations were not affected; (3) a remarkable evolution of adoptive cell therapy is represented by NK cells engineered with CAR-targeting tumor antigens (CAR-NK). CAR-NK cells complement CAR-T cells as they do not cause GvHD and may be obtained from unrelated donors. Accordingly, CAR-NK cells may represent an “off-the-shelf” tool, readily available for effective tumor therapy; (4) the efficacy of adoptive cell therapy in cancer is also witnessed by the αβT cell- and B cell-depleted haploidentical HSC transplantation in which the infusion of donor NK cells and γδT cells, together with HSC, sharply reduces leukemia relapses and infections; (5) a true revolution in tumor therapy is the use of mAbs targeting checkpoint inhibitors including PD-1, CTLA-4, the HLA class I-specific KIR, and NKG2A. Since PD-1 is expressed not only by tumor-associated T cells but also by NK cells, its blocking might unleash NK cells playing a crucial effector role against HLA class I-deficient tumors that are undetectable by T cells.

An Historical Overview: The Discovery of How NK Cells Can Kill Enemies, Recruit Defense Troops, and More.

Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity characterized by the unique ability of killing tumor and virally infected cells without any prior priming and expansion of specific clones. The “missing-self” theory, proposed by Klas Karre, the seminal discovery of the first prototypic HLA class I-specific inhibitory receptors, and, later, of the Natural Cytotoxicity Receptors (NCRs) by Alessandro Moretta, provided the bases to understand the puzzling behavior of NK cells. Actually, those discoveries proved crucial also for many of the achievements that, along the years, have contributed to the modern view of these cells. Indeed, NK cells, besides killing susceptible targets, are now known to functionally interact with different immune cells, sense pathogens using TLR, adapt their responses to the local environment, and, even, mount a sort of immunological memory. In this review, we will specifically focus on the main activating NK receptors and on their crucial role in the ever-increasing number of functions assigned to NK cells and other innate lymphoid cells (ILCs).

Natural killer cells: From surface receptors to the cure of high-risk leukemia (Ceppellini Lecture).

Natural killer (NK) cells are innate immune effector cells involved in the first line of defense against viral infections and malignancies. In the last three decades, the identification of HLA class I‐specific inhibitory killer immunoglobulin‐like receptors (KIR) and of the main activating receptors has strongly improved our understanding of the mechanisms regulating NK cell functions. The increased knowledge on how NK cells discriminate healthy cells from damaged cells has made it possible to transfer basic research notions to clinical applications. Of particular relevance is the strong NK‐mediated anti‐leukemia effect in haploidentical hematopoietic stem cell transplantation to cure high‐risk leukemia.

Human NK cells: surface receptors, inhibitory checkpoints, and translational applications.

NK cells play important roles in innate defenses against viruses and in the control of tumor growth and metastasis. The regulation/induction of NK cell function is mediated by an array of activating or inhibitory surface receptors. In humans, major activating receptors involved in target cell killing are the natural cytotoxicity receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or expressed de novo upon cell stress, viral infection, or tumor transformation. The HLA-class I-specific inhibitory receptors, including KIRs recognizing HLA-class I allotypic determinants and CD94/NKG2A recognizing the class-Ib HLA-E, constitute a fail-safe mechanism to avoid unwanted NK-mediated damage to healthy cells. Other receptors such as PD-1, primarily expressed by activated T lymphocytes, are important inhibitory checkpoints of immune responses that ensure T-cell tolerance. PD-1 also may be expressed by NK cells in cancer patients. Since PD-1 ligand (PD-L1) may be expressed by different tumors, PD-1/PD-L1 interactions inactivate both T and NK cells. Thus, the reliable evaluation of PD-L1 expression in tumors has become a major issue to select patients who may benefit from therapy with mAbs disrupting PD-1/PD-L1 interactions. Recently, NKG2A was revealed to be an important checkpoint controlling both NK and T-cell activation. Since most tumors express HLA-E, mAbs targeting NKG2A has been used alone or in combination with other therapeutic mAbs targeting PD-1 or tumor antigens (e.g., EGFR), with encouraging results. The translational value of NK cells and their receptors is evidenced by the extraordinary therapeutic success of haploidentical HSCT to cure otherwise fatal high-risk leukemias.

P057 : INFLUENCE OF ACTIVATING AND INHIBITORY KILLER IMMUNOGLOBULIN-LIKE RECEPTOR GENES ON PREDISPOSITION TO LYMPHOCYTIC LEUKEMIAS

Often referred to as the ‘third lymphocytes’, Natural Killer (NK) cells are critical to early control of viral infections and immune-surveillance of tumors and leukemias. Regulation of NK cell responses is mediated by one or more HLA class-I specific inhibitory receptors, (chiefly, of the Killer immunoglobulin-like receptor (KIR) family), in a way that it can only be triggered when the inhibitory signals are either diminished or are overridden by activating signals. Given the remarkable genetic diversity demonstrated by the KIR gene cluster, it is hypothesized that different KIR gene repertoires characterized by differences in activating and inhibitory gene content could be associated with differential capacities of NK cells (and certain T cell subsets) to eliminate leukemic target cells. A total of 218 subjects including healthy individuals ( n = 124) and patients of myelogenous ( n = 62) and lymphocytic ( n = 32) leukemia respectively were analyzed for KIR gene repertoire distribution using Luminex™ based assay. Differences in average incidences of individual KIR genes and haplotypes among all three groups were tested for significance. Significantly reduced incidences of activating KIR2DS3 ( p = 0.009; OR = 5.7) and KIR3DS1 ( p = 0.01; OR = 3.1) were observed in patients suffering from lymphoid malignancies and not in patients suffering from myeloid malignancies. KIR gene repertoires containing ⩽ 1 activating KIR were significantly more often represented by the lymphocytic leukemia patients ( p = 0.03; OR = 3.5) as compared to controls who most often presented ⩾5 activating KIR. This finding supports the hypothesis that an absence of appropriate activation signals may provide lymphocytic leukemias an escape mechanism from immune surveillance by NK cells.

At the Bench: Preclinical rationale for exploiting NK cells and γδ T lymphocytes for the treatment of high-risk leukemias

NK cells and γδ T lymphocytes display potent cytolytic activity against leukemias and CMV-infected cells and are thus, promising immune effector cells in the context of allo-HSCT. NK cells express HLA class I-specific inhibitory receptors and preferentially kill HLA class I(low) tumors or virus-infected cells. Killing occurs upon engagement of activating NKRs with ligands that are up-regulated on tumors and infected cells. A similar activating receptor/ligand interaction strategy is used by γδ T cells, which in addition, use their TCRs for recognition of phosphorylated antigens and still largely undefined ligands on tumor cells. In the haploidentical allo-HSCT setting, alloreactive NK cells, derived from donor HSCs, can exert potent antileukemia activity and kill residual patient DCs and T cells, thus preventing GvHD and graft rejection. However, generation of KIR(+) alloreactive NK cells from HSCs requires many weeks, during which leukemia relapses, and life-threatening infections may occur. Importantly, mature NK cells and γδ T cells can control certain infectious agents efficiently, in particular, limit CMV reactivation, and infusion of such donor cells at the time of HSCT has been implemented. Development of novel, cell-based immunotherapies, allowing improved trafficking and better targeting, will endow NK cells and γδ T lymphocytes with enhanced anti-tumor activity, also making them key reagents for therapies against solid tumors. The clinical aspects of using NK cells and γδ T lymphocytes against hematological malignancies, including the allo-HSCT context, are reviewed in the related side-by-side paper by Locatelli and colleagues [1].

NK cells from malignant pleural effusions are not anergic but produce cytokines and display strong antitumor activity on short‐term IL‐2 activation

NK cells are a major component of innate immunity and exert a potent antitumor effect both in vitro and in vivo. However, NK cells infiltrating solid tumors have been shown to display severely impaired functional capabilities. In this study, we analyzed NK cells present in pleural effusions (PEs) of patients with primary or metastatic tumors of different origin, including mesothelioma and lung, breast, colon, gastric, bladder, and uterus carcinoma. In all instances, freshly isolated PE-NK cells displayed a CD56(bright) phenotype and expressed normal levels of both activating receptors and HLA class I-specific inhibitory receptors. In addition, they rapidly released large amounts of IFN-γ and TNF-α after stimulation. Upon culture in IL-2, they acquired a potent cytolytic activity against both allogeneic and autologous tumor cells. Tumor cell lysis was primarily mediated by NKG2D and NKp30 and partially by NKp46 and DNAM-1, in agreement with the expression of the corresponding ligands on tumor cells. The finding that PE-NK cells are not functionally impaired and that they can efficiently kill tumor cells upon short-term IL-2 activation may offer important clues for the development of novel approaches in tumor immunotherapy.

Alloreactive natural killer cells in targeting high-risk leukaemias

Natural killer (NK) cells express surface receptors that regulate potent effector functions, such as cytolytic activity and release of cytokines, which play a central role in inflammatory response and immunoregulation....

NK cell-mediated immune response against cancer

The past fifteen years have witnessed great progress in our undestanding of how natural killer (NK) cells function, their role in innate defenses and their possible exploitation in therapy. This contribution traces the major advances in these formerly mysterious cells, from the first discovery of HLA-class I-specific inhibitory receptors to a more recent major breakgthrough that highlighted important perspectives and major expectations regarding the cure of life-threatening leukemias. The key role of "alloreactive" NK cells in eradicating acute myeloid leukemias and in preventing both graft rejection and graft-versus-host disease, led to a true revolution in bone marrow transplantation. Thus, it is now possible to search for appropriate HLA class I mismatches to set NK cells in action to cure high risk leukemias.

STAT5A Overexpression Enhances Killer Immunoglobulin Receptor (KIR) Expression in Developing NK Cells and Is Associated with a Loss of Reverse Transcription from the Proximal KIR Promoter.

Down-regulation of HLA-class I molecules is commonly observed in virally infected and malignantly transformed cells and can contribute to the ability of these cells to escape recognition by the adaptive immune system. NK cells are able to detect the loss of even single HLA alleles on potential target cells through their clonally distributed HLA-class I-specific inhibitory receptors (KIR). While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides in areas surrounding the transcription initiation region, the mechanisms that regulate variegated KIR expression are largely unknown. The manipulation of KIR expression is of considerable interest due to the widely reported correlation between KIR ligand status and patient survival in hematopoietic stem cell transplant settings. Because the transcription factor STAT5 is activated downstream of the BCR/ABL oncogene, which we have previously shown to substantially augment KIR expression levels in primary NK cells, we hypothesized that STAT5 could directly influence KIR expression. To test this hypothesis, we purified CD34+ hematopoietic progenitor cells from umbilical cord blood and retrovirally transduced these cells with either a control murine stem cell virus (MSCV) vector encoding eGFP alone or an MSCV vector encoding both eGFP and a constitutively active form of STAT5A, termed STAT5A 1*6. CD34/eGFP-positive cells were then purified and cultured on the EL08-D1 stroma line, known to support NK cell development. After a period of 28 days in culture, 6.25±2.73% of the NK cells in eGFP control cultures were KIR-positive compared with 29.3±4.27% of STAT5A 1*6 transductants. To more closely examine individual KIR expression at the transcriptional level, we carried out a quantitative RT-PCR analysis with probe sets previously validated for amplification of individual KIR (Cooley et al., Blood). We observed a general increase in the mRNA expression levels of both inhibitory and activating KIR in STAT5A 1*6-transduced cells. In order to investigate the mechanism underlying the increased KIR expression observed in the STAT5A 1*6-transductants, we performed an RT-PCR specific for the reverse transcript originating from the proximal promoter of the KIR3DL1 gene. In a model proposed by Anderson et al., reverse transcription from the bidirectional proximal promoter of variegated KIR genes may be responsible for the silencing of the KIR locus through the production of dsRNA, which then induces DNA methylation in a siRNA-dependent manner. Our RT-PCR results from multiple donors clearly show an absence of reverse proximal transcript in cells transduced with the STAT5A 1*6 construct in contrast to high levels of transcript in eGFP controls. These results provide evidence for a role of STAT5A in the induction of KIR expression and support a model of KIR regulation involving intergenic transcription and possibly siRNA-mediated gene silencing. A better understanding of how to manipulate these KIR expression patterns may be of benefit to exploit the potential of NK cell therapy to improve transplant outcomes.

Identification of effector-memory CMV-specific T lymphocytes that kill CMV-infected target cells in an HLA-E-restricted fashion

HLA-E-restricted T cells represent a minor cytolytic T lymphocyte (CTL) population characterized by the surface expression of HLA class I-specific inhibitory receptors and by the capability of killing a large panel of allogeneic target cells (therefore named NK-CTL). Here we show that this subset of T cells is present in a sizeable fraction in the peripheral blood of human cytomegalovirus (HCMV)-seropositive healthy individuals. We provide evidence that NK-CTL recognize in an HLA-E-restricted fashion a naturally processed CMV-derived peptide in the transporter associated with antigen processing (TAP)-2(-/-) UL40+ RMA-S cell transfectants. Moreover, we show that they recognize and kill HCMV-infected target cells. NK-CTL are characterized by the CD8beta(dull) (CD45RA+)(CD28-)(CD27-)(CCR7-)(CD56+) surface phenotype, thus suggesting that they belong to the effector-memory cell compartment. Consistent with the effector-memory phenotype, they promptly produce IFN-gamma, but not IL-2, upon interaction with the specific HCMV UL40-derived peptide. Our data suggest that HLA-E-restricted CTL may represent an additional effector cell type involved in defenses against HCMV, a virus which escapes the control exerted by conventional CTL or NK cells.

Tm1 cells, a subset of T cells with NK cell and memory CD8 T‐cell features

Tolerance of NK cell toward self is ensured in part by inhibitory receptors for MHC class I receptors. These receptors include killer cell Ig-like receptors (KIRs) and leukocyte Ig-like receptors (LIRs) in humans, Ly49 molecules in mice, as well as CD94/NKG2A heterodimers in both species. These receptors can also be expressed on T cells. Whereas CD94/NKG2A heterodimers can be readily expressed after T-cell activation, the induction of KIR or LIR on human T cells, as well of Ly49 molecules on mouse T cells, appears more tightly regulated. We will present data that characterize T memory type 1 cells (Tm1 cells), the subset of CD8+ T cells that express KIR/LIR in humans or Ly49 in mice. Nakajima T, Goek O, Zhang X, et al. De novo expression of killer immunoglobulin-like receptors and signaling proteins regulates the cytotoxic function of CD4 T cells in acute coronary syndromes. Circ Res [Epub ahead of print]. Induction of KIR on CD4+ T cells. Such expansions occur in acute coronary syndromes as well as in rheumatoid arthritis patients. Wechsler J, Bagot M, Nikolova M, et al. Killer cell immunoglobulin-like receptor expression delineates in situ Sezary syndrome lymphocytes. J Pathol 2003: 199 (1): 77–83. Sezary cells selectively express KIR3DL2. This article reviews these very important sets of information. Romagnani C, Pietra G, Falco M, et al. Identification of HLA-E-specific 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 (17): 11328–11333. First complete description of HLA-E-restricted T cells is KIR+ T cells. Ugolini S, Arpin C, Anfossi N, et al. Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells. Nat Immunol 2001: 2 (5): 430–435. First demonstration that KIR engagement can participate in the survival of Tm1 cells. Vely F, Peyrat M, Couedel C, 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 (4): 2487–2494. Young N T, Uhrberg M, Phillips J H, Lanier L L, Parham P. Differential expression of leukocyte receptor complex-encoded Ig-like receptors correlates with the transition from effector to memory CTL. J Immunol 2001: 166 (6): 3933–3941. These two papers demonstrate that KIR are acquired after VDJ recombination. Andre P, Brunet C, Guia S, et al. Differential regulation of killer cell Ig-like receptors and CD94 lectin-like dimers on NK and T lymphocytes from HIV-1-infected individuals. Eur J Immunol 1999: 29 (4): 1076–1085. This paper first shows that the mechanisms involved in the regulation of CD94/NKG2A and KIR expression in vivo are different. Bakker A B, Phillips J H, Figdor C G, Lanier L L. Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, γδ T cells, and antigen-specific CTL. J Immunol 1998: 160 (11): 5239–5245. De Maria A, Ferraris A, Guastella M, et al. Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: impairment of specific cytolytic functions. Proc Natl Acad Sci USA 1997: 94 (19): 10285–10288. Together with several other publications from several groups, these two articles show that KIR engagement can inhibit T-cell effector function.

Expression of p58.2 or CD94/NKG2A inhibitory receptors in an NK-like cell line, YTINDY, leads to HLA Class I-mediated inhibition of cytotoxicity in the p58.2- but not the CD94/NKG2A-expressing transfectant

Natural killer cytotoxicity is down-regulated by HLA Class I-specific inhibitory receptors classified as killer inhibitory receptors (KIRs) or C-type lectins. The regulation of their inhibitory signaling pathways is not completely understood. The YTINDY NK-like cell line was transfected to express p58.2 KIR (YT/C143 transfectant) or CD94/NKG2A C-type lectin (YT/CD94 transfectant); and YT/C143, but not YT/CD94, cytotoxicity was down-regulated by Class I. YT/C143 and YT/CD94 expressed equally low p56 lck levels, suggesting that p56 lck is not absolutely required for p58.2 signaling but may be required for CD94/NKG2A signaling. Lower SHP-1 levels and activity were observed in YT/CD94 compared to YT/C143. However, increasing SHP-1 to equivalent levels in YT/C143 did not restore inhibition in YT/CD94. Our results suggest that the combination of low p56 lck and SHP-1 levels may be responsible for the absent inhibitory signal in YT/CD94. In addition, the possible expression of CD94/NKG2C activating receptor may override inhibitory signals transduced through CD94/NKG2A.

Identification of HLA-E-specific alloreactive T lymphocytes: a cell subset that undergoes preferential expansion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells.

Previous studies showed that a subset of CD8(+) cytolytic T lymphocytes expressing HLA class I-specific natural killer inhibitory receptors (iNKR) is characterized by the ability to recognize HLA-E and to mediate T cell receptor-dependent killing of different NK cell-susceptible tumor target cells. In this study, we show that this CD8(+) T cell subset can also undergo extensive proliferation in mixed lymphocyte cultures in response to allogeneic cells. Analysis of their cytolytic activity revealed a broad specificity against a panel of allogeneic phytohemagglutinin-induced blasts derived from HLA-unmatched donors. On the other hand, autologous and certain allogeneic phytohemagglutinin blasts were resistant to lysis. We used as target cells the transporter associated with antigen processing (TAP)-2(-/-) murine RMA-S cell line cotransfected with beta2-microglobulin and HLA-E*01033. On loading these cells with different HLA-E-binding peptides derived either from HLA class I leader sequences or viral proteins, we could show that HLA-E-specific cytolytic T lymphocytes recognized many, but not all, peptides analyzed. These data suggest that these cells may recognize, on allogeneic cells, HLA-E with peptides that are not present in the host of origin. In addition to their ability to proliferate in response to allogeneic stimulation and to lyse, most allogeneic cells may have important implications in transplantation and in antitumor immune responses.

Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation.

In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34(+) Lin(-) cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor.

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR alpha/beta-mediated recognition.

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8(+) CTL has been shown to lyse certain natural killer (NK)-susceptible target cells via still undefined mechanism(s). These CD8(+) T cells, hereafter referred to as NK-CTL, are frequently composed of cells expressing one single TCR Vbeta expansion (different in different individuals), display a memory phenotype and express HLA class I-specific inhibitory NK receptors. Here we show that cell populations or clones of NK-CTL isolated from three healthy donors homogeneously expressed Vbeta16, Vbeta9 and Vbeta3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vbeta16(+) cells of donor 1 displayed identical TCR Vbeta and Valpha rearrangements, thus suggesting a substantial monoclonality of the NK-CTL subset analyzed. NK-CTL lysed a number of NK-susceptible tumor target cells with the exception of those characterized by beta2-microglobulin (beta2m) deficiency. However, the latter targets became susceptible to lysis upon beta2m transfection. Using monoclonal antibodies specific for the relevant TCR Vbeta or beta2m we provide evidence suggesting that target cell lysis by NK-CTL is mediated by the TCR itself upon recognition of beta2m-associated proteins. The cellular distribution of the potential beta2m-associated proteins in susceptible target cells suggested, as a likely candidate for TCR-mediated recognition, the non-classical HLA-E molecule. The use, as target cells, of the murine TAP2-deficient RMA-S cells, either untransfected or transfected with HLA-E, and loaded with an appropriate HLA-E-binding peptide, provided the direct demonstration that HLA-E represents a ligand recognized by the TCR expressed by NK-CTL. This is the first evidence that human TCR alpha/beta can recognize HLA-E molecules, thus revealing a novel type of TCR-mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.

Human natural killer cell receptors and co-receptors.

In the absence of sufficient signaling by their HLA class I-specific inhibitory receptors, human natural killer (NK) cells become activated and display potent cytotoxicity against cells that are either HLA class I negative or deficient. This indicates that the NK receptors responsible for the induction of cytotoxicity recognize ligands on target cells different from HLA class I molecules. On this basis, the process of NK-cell triggering can be considered as a mainly non-MHC-restricted mechanism. The recent identification of a group of NK-specific triggering surface molecules has allowed a first series of pioneering studies on the functional/molecular characteristics of such receptors. The first three members of a receptor family that has been termed natural cytotoxicity receptors (NCR) are represented by NKp46, NKp44 and NKp30. These receptors are strictly confined to NK cells, and their engagement induces a strong activation of NK-mediated cytolysis. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various target cells. Importantly, mAb-mediated blocking of these receptors has been shown to suppress cytotoxicity against most NK-susceptible target cells. However, the process of NK-cell triggering during target cell lysis may also depend on the concerted action of NCR and other triggering receptors, such as NKG2D, or surface molecules, including 2B4 and NKp80, that appear to function as co-receptors rather than as true receptors. Notably, a dysfunction of 2B4 has been associated with a severe form of immunodeficiency termed X-linked lymphoproliferative disease. Future studies will clarify whether also the altered expression and/or function of other NK-triggering molecules may represent a possible cause of immunological disorders.

Differential disappearance of inhibitory natural killer cell receptors during HAART and possible impairment of HIV-1-specific CD8 cytotoxic T lymphocytes.

Highly active antiretroviral therapy (HAART) is associated with a decrease in viral replication to undetectable levels and with an increase in CD4 T lymphocytes. Residual HIV-1 replication occurs together with incomplete recovery of cytotoxic CD8 T lymphocyte (CTL) numbers and function. We sought to determine whether expression of HLA class I-specific inhibitory natural killer receptors (iNKR) on the CTL of patients who had been treated successfully with HAART for 24 months could be involved, at least in part, in residual CTL functional inhibition. Two-colour cytofluorometry was used to analyse the expression of six different iNKR including p58.1, p58.2, p70, p140, CD94/NKG2A and LIR1/ILT2 on the CD3, CD8 lymphocytes of eight patients with successful long-term suppression of viral replication before and after 3, 6 and 24 months of HAART. Healthy subjects were analysed as controls. HIV-1-specific cytotoxic activity was determined after 24 months of HAART in the presence and absence of iNKR-masking. No significant reduction of iNKR expression on CD8 T cells was observed by 6 months. Expression of p70 and p140 was inversely correlated with the increasing CD4 numbers. After 24 months CD8 T-lymphocytes expressing p58.1, p58.2, p70, p140 and CD94/NKG2A returned to levels indistinguishable from those of the healthy controls. A significantly increased proportion of CD8 CTL still expressed LIR1/ILT2, a receptor with broad HLA-class I specificity. Functional analysis of freshly separated cells revealed that the disruption of the interaction between LIR1/ILT2 and HLA-class I could partly restore HIV-1-specific lysis. A decrease in CD3CD8iNKR cells is observed beyond 6 months of HAART. In some patients functional impairment due to LIR1/ILT2 expression may persist even after 24 months of successful HAART.