Romana Conte

Transforming growth factor β1 inhibits expression of NKp30 and NKG2D receptors: Consequences for the NK-mediated killing of dendritic cells

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor β1 (TGFβ1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFβ1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFβ1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFβ1-producing dendritic cells may acquire resistance to the NK-mediated attack.

CD56brightCD16− Killer Ig-Like Receptor− NK Cells Display Longer Telomeres and Acquire Features of CD56dim NK Cells upon Activation

Human NK cells can be divided into CD56dimCD16+ killer Ig-like receptors (KIR)+/− and CD56brightCD16− KIR− subsets that have been characterized extensively regarding their different functions, phenotype, and tissue localization. Nonetheless, the developmental relationship between these two NK cell subsets remains controversial. We report that, upon cytokine activation, peripheral blood (PB)-CD56bright NK cells mainly gain the signature of CD56dim NK cells. Remarkably, KIR can be induced not only on CD56bright, but also on CD56dim KIR− NK cells, and their expression correlates with lower proliferative response. In addition, we demonstrate for the first time that PB-CD56dim display shorter telomeres than PB- and lymph node (LN)-derived CD56bright NK cells. Along this line, although human NK cells collected from nonreactive LN display almost no KIR and CD16 expression, NK cells derived from highly reactive LN, efferent lymph, and PB express significant amounts of KIR and CD16, implying that CD56bright NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR+CD16+ NK cells. Altogether, our results suggest that CD56brightCD16− KIR− and CD56dimCD16+KIR+/− NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs can be sites of NK cell final maturation and self-tolerance acquisition during immune reaction.

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.

p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig‐SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig‐SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig‐like domains and a 115‐amino acid cytoplasmic tail containing a single immuno‐receptor tyrosine‐based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig‐SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR+ T cell clones. cl.15.212 cDNA codes for a membrane‐bound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212‐encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA‐G1, ‐A3, ‐B46 alleles and weakly to ‐B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA‐A2, ‐B51, ‐Cw3 or ‐Cw4. The binding specificity of soluble p49‐Fc was confirmed by competition experiments using an anti‐HLA class I‐specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.

Melanoma Cells Inhibit Natural Killer Cell Function by Modulating the Expression of Activating Receptors and Cytolytic Activity

Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.

CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells

Natural killer (NK) cells are the main lymphoid population in the maternal decidua during the first trimester of pregnancy. Decidual NK (dNK) cells display a unique functional profile and play a key role in promoting tissue remodeling, neoangiogenesis, and immune modulation. However, little information exists on their origin and development. Here we discovered CD34+ hematopoietic precursors in human decidua (dCD34+). We show that dCD34+ cells differ from cord blood- or peripheral blood-derived CD34+ precursors. The expression of IL-15/IL-2 receptor common β-chain (CD122), IL-7 receptor α-chain (CD127), and mRNA for E4BP4 and ID2 transcription factors suggested that dCD34+ cells are committed to the NK cell lineage. Moreover, they could undergo in vitro differentiation into functional (i.e., IL-8– and IL-22–producing) CD56brightCD16−KIR+/− NK cells in the presence of growth factors or even upon coculture with decidual stromal cells. Their NK cell commitment was further supported by the failure to undergo myeloid differentiation in the presence of GM-CSF. Our findings strongly suggest that decidual NK cells may directly derive from CD34+ cell precursors present in the decidua upon specific cellular interactions with components of the decidual microenvironment.

The Three-Dimensional Structure of the Human NK Cell Receptor NKp44, a Triggering Partner in Natural Cytotoxicity

Natural killer (NK) cells direct cytotoxicity against tumor or virally infected cells. NK cell activation depends on a fine balance between inhibitory and activating receptors. NKp44 is a cytotoxicity activating receptor composed of one Ig-like extracellular domain, a transmembrane segment, and a cytoplasmic domain. The 2.2 A crystal structure shows that the NKp44 Ig domain forms a saddle-shaped dimer, where a charged surface groove protrudes from the core structure in each subunit. NKp44 Ig domain disulfide bridge topology defines a new Ig structural subfamily. The data presented are a first step toward understanding the molecular basis for ligand recognition by natural cytotoxicity receptors, whose key role in the immune system is established, but whose cellular ligands are still elusive.

NCR + ILC3 concentrate in human lung cancer and associate with intratumoral lymphoid structures

Tertiary lymphoid structures (TLSs) are a common finding in non-small cell lung cancer (NSCLC) and are predictors of favourable clinical outcome. Here we show that NCR(+) innate lymphoid cell (ILC)-3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumour-associated TLSs. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, on activation, produces IL-22, TNF-α, IL-8 and IL-2, and activates endothelial cells. Tumour NCR(+)ILC3 may interact with both lung tumour cells and tumour-associated fibroblasts, resulting in the release of cytokines primarily on engagement of the NKp44-activating receptor. In patients, NCR(+)ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumour stages and their presence correlate with the density of intratumoral TLSs. Our results indicate that NCR(+)ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumour-associated TLSs.

Molecular and Functional Characterization of NKG2D, NKp80, and NKG2C Triggering NK Cell Receptors in Rhesus and Cynomolgus Macaques: Monitoring of NK Cell Function during Simian HIV Infection

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.

Human NK cells at early stages of differentiation produce CXCL8 and express CD161 molecule that functions as an activating receptor

Human natural killer (NK) cell development is a step-by-step process characterized by phenotypically identified stages. CD161 is a marker informative of the NK cell lineage commitment, whereas CD56, CD117, and CD94/NKG2A contribute to define discrete differentiation stages. In cells undergoing in vitro differentiation from CD34(+) umbilical cord blood (UCB) progenitors, LFA-1 expression allowed to discriminate between immature noncytolytic CD161(+)CD56(+)LFA-1(-) and more differentiated cytolytic CD161(+)CD56(+)LFA-1(+) NK cells. CD161(+)CD56(+)LFA-1(-) NK cells produce large amounts of CXCL8 after phorbol myristate acetate (PMA) or cytokine treatment. Remarkably, CXCL8 mRNA expression was also detected in fresh stage III immature NK cells isolated from tonsils and these cells expressed CXCL8 protein on PMA stimulation. Within in vitro UCB-derived CD161(+)CD56(+)LFA-1(-) NK cells, CXCL8 release was also induced on antibody-mediated cross-linking of NKp44 and CD161. Such unexpected activating function of CD161 was confined to the CD161(+)CD56(+)LFA-1(-) subset, because it did not induce cytokine release or CD107a expression in CD161(+)CD56(+)LFA-1(+) cells or in mature peripheral blood NK cells. Anti-CXCL8 neutralizing antibody induced a partial inhibition of NK cell differentiation, which suggests a regulatory role of CXCL8 during early NK cell differentiation. Altogether, these data provide novel information that may offer clues to optimize NK cell maturation in hematopoietic stem cell transplantation.