Silvia Parolini

Human natural killer cell receptors and co‐receptors

Summary: 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.

2B4 functions as a co‐receptor in human NK cell activation

Natural cytotoxicity receptors (NKp46, NKp44 and NKp30) play a predominant role in human NK cell triggering during natural cytotoxicity. Human 2B4 also induced NK cell activation in redirected killing assays using anti‐2B4 monoclonal antibodies (mAb) and murine targets. Since this effect was confined to a fraction of NK cells, this suggested a functional heterogeneity of 2B4 molecules. Here we show that activation via 2B4 in redirected killing against murine targets is strictly dependent upon the engagement of NKp46 by murine ligand (s) on target cells. Thus, NK cell clones expressing high surface density of NKp46 (NKp46bright) were triggered by anti‐2B4 mAb, whereas NKp46dull clones were not although they expressed a comparable surface density of 2B4. mAb‐mediated modulation of NKp46 molecules in NKp46bright clones had no effect on the expression of 2B4 while it rendered cells unresponsive to anti‐2B4 mAb. Finally, anti‐2B4 mAb could induce NK cell triggering in NKp46dull clones provided that suboptimal doses of anti‐NKp44 or anti‐CD16 mAb were added to the redirected killing assay. These results indicate that differences in responses do not reflect a functional heterogeneity of 2B4 but rather depend on the co‐engagement of triggering receptors.

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.

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34+ Lin– hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect ofdifferent cytokines. In this study we show that IL‐21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34+ Lin– cells isolated from human cord blood supplemented with IL‐15, Flt3‐L and SCF. After 25 days of culture, 50% of CD56+ cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectablein developing NK cells cultured in the absence of IL‐21. Remarkably, similar to mature NK cells all these markers were included in the CD56dim cell fraction, while the CD56bright population was only composed of CD94/NKG2A– and CD94/NKG2A+ cells. Thus, IL‐21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.

Identification of NKp80, a novel triggering molecule expressed by human NK cells

The ability of NK cells to kill a wide range of tumor or virally infected target cells as well as normal allogeneic T cell blasts appears to depend upon the concerted action of multiple triggering NK receptors. In this study, using two specific monoclonal antibodies [(mAb) MA152 and LAP171], we identified a triggering NK receptor expressed at the cell surface as a dimer of approximately 80 kDa (NKp80). NKp80 is expressed by virtually all fresh or activated NK cells and by a minor subset of T cells characterized by the CD56 surface antigen. NKp80 surface expression was also detected in all CD3– and in 6 / 10 CD3+ large granular lymphocyte expansions derived from patients with lymphoproliferative disease of granular lymphocytes. In polyclonal NK cells, mAb‐mediated cross‐linking of NKp80 resulted in induction of cytolytic activity and Ca2 + mobilization. A marked heterogeneity existed in the magnitude of the cytolytic responses of different NK cell clones to anti‐NKp80 mAb. This heterogeneity correlated with the surface density of NKp46 molecules expressed by different NK clones. The mAb‐mediated masking of NKp80 led to a partial inhibition of the NK‐mediated lysis of appropriate allogeneic phytohemagglutinin‐induced T cell blasts, while it had no effect on the lysis of different tumor target cells, including T cell leukemia cells. These data suggest that NKp80 recognizes a ligand on normal T cells that may be down‐regulated during tumor transformation. Molecular cloning of the cDNA coding for NKp80 revealed a type II transmembrane molecule of 231 amino acids identical to the putative protein encoded by a recently identified cDNA termed KLRF1.

IL-12 or IL-4 Prime Human NK Cells to Mediate Functionally Divergent Interactions with Dendritic Cells or Tumors

In the course of inflammatory responses in peripheral tissues, NK cells may be exposed to cytokines such as IL-12 and IL-4 released by other cell types that may influence their functional activities. In the present study we comparatively analyzed purified human peripheral blood NK cells that had been exposed to either IL-12 or IL-4 during short (overnight) incubation. We show that although IL-12-cultured NK cells produced abundant IFN-γ, TNF-α, and GM-CSF in response to stimuli acting on the NKp46-activating receptor, IL-4-cultured NK cells did not release detectable levels of these cytokines. In contrast, IL-4-cultured NK cells produced significant levels of TNF-α and GM-CSF only when stimulated with PMA and ionomycin. In no instance could the production of IL-5 and IL-13 be detected. Importantly, IL-12-cultured, but not IL-4-cultured, NK cells displayed strong cytolytic activity against various tumor cells or immature dendritic cells (DCs). Moreover, only NK cells that had been cultured in IL-12 were able to induce substantial DC maturation. Our data suggest that NK cells exposed to IL-12 for a time interval compatible with in vivo responses may favor the selection of appropriate mature DCs for subsequent Th1 cell priming in secondary lymphoid organs. On the contrary, NK cells exposed to IL-4 do not exert DC selection, may impair efficient Th1 priming, and favor either tolerogenic or Th2-type responses.

Basic fibroblast growth factor overexpression in endothelial cells: an autocrine mechanism for angiogenesis and angioproliferative diseases

Basic fibroblast growth factor (bFGF) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases. The ultimate significance of this observation is poorly understood. We have investigated the biological consequences of endothelial cell activation by endogenous bFGF in a mouse aortic endothelial cell line stably transfected with a retroviral expression vector harboring a human bFGF cDNA. Selected clones expressing M(r) 24,000, M(r) 22,000, and/or M(r) 18,000 bFGF isoforms were characterized by a transformed morphology and an increased saturation density. bFGF transfectants showed invasive behavior and sprouting activity in three-dimensional fibrin gels and formed a complex network of branching cord-like structures connecting foci of infiltrating cells when seeded on laminin-rich basement membrane matrix (Matrigel). The invasive and morphogenetic behavior was prevented by anti-bFGF antibody, revealing the autocrine modality of the process. The biological consequences of this autocrine activation were investigated in vivo. bFGF-transfected cells gave rise to highly vascularized lesions resembling Kaposis sarcoma when injected in nude mice and induced angiogenesis in avascular rabbit cornea. When injected into the allantoic sac of the chick embryo, they caused an increase in vascular density and formation of hemangiomas in the chorioallantoic membrane. In conclusion, bFGF-overexpressing endothelial cells acquired an angiogenic phenotype and recruit quiescent endothelium originating angioproliferative lesions in vivo. These findings demonstrate that bFGF overexpression exerts an autocrine role for endothelial cells and support the notion that tumor neovascularization and angioproliferative diseases can be triggered by stimuli that induce vascular endothelium to produce its own autocrine factor(s).

Involvement of natural cytotoxicity receptors in human natural killer cell-mediated lysis of neuroblastoma and glioblastoma cell lines

The surface receptors involved in natural killer (NK) cell triggering during the process of target cell lysis have been at least in part identified. These are members of a novel family of receptors that has been termed natural cytotoxicity receptors (NCR). The first three members of this emerging group of receptors are the NKp46, NKp44 and NKp30 molecules that all belong to the immunoglobulin superfamily. Blocking of these receptors inhibits NK-mediated cytotoxicity against a wide variety of tumor target cells. In the present study, we show that these NCR are also involved in NK-mediated killing of tumor cells of neural origin. Glioblastoma and neuroblastoma target cells were efficiently killed by all NK clones analyzed since little protection from NK lysis was mediated by HLA class I molecules. Blocking of one or another NCR inhibited cytotoxicity; however, optimal inhibition was only observed when the three receptors were blocked simultaneously. A sharp difference in cytotoxicity against neural tumors was demonstrated between NCR(bright) and NCR(dull) NK clones, further supporting the notion that NCR play a critical role in the induction of cytotoxicity against tumor target cells of different histotype. Finally, our data also indicate that CD16 does not function as a triggering receptor involved in lysis of neural tumors since no difference in cytotoxicity could be substantiated between CD16(+) and CD16(-) NK clones and no correlation could be detected between the NCR(bright)/NCR(dull) phenotype and CD16 expression.

A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.

A homozygous mutation that gave rise to a stop codon in the WIPF1 gene resulted in WASP protein destabilization and in symptoms resembling those of Wiskott-Aldrich syndrome

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB‐A and NKp80, function as co‐receptors. As reported in the present study, during an attempt to identify novel NK receptors or co‐receptors, we found that CD59 functions as a co‐receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co‐receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)‐linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59‐dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb‐mediated modulation of NKp46 resulted in markedly decreased responses to anti‐CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3ζ chains associated with these NCR, but not those associated with CD16. Thus, CD59‐mediated costimulation of NK cells requires direct physical interaction of this GPI‐linked protein with primary triggeringNK receptors.