Frédéric Vély

Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A : association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases

Subsets of T and natural killer (NK) lymphocytes express the CD94‐NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94‐NKG2A heterodimer inhibits both antigen‐driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma‐specific human T cell clones. Similarly, CD16‐mediated NK cell cytotoxicity is extinguished by cross‐linking of the CD94‐NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine‐based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP‐1 and SHP‐2, but not with the polyinositol phosphatase SHIP. Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho‐ITIM interact directly with the SH2 domains of SHP‐1 and SHP‐2 with a high affinity. Engagement of the CD94‐NKG2A heterodimer therefore appears as a protein‐tyrosine phosphatase‐based strategy that negatively regulates both antigen‐induced T cell response and antibody‐induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.

Tuning of Natural Killer Cell Reactivity by NKp46 and Helios Calibrates T Cell Responses

Natural Killer Controls Cytolytic natural killer (NK) cells participate in both antimicrobial and antitumor immunity. Their responsiveness is tuned through signals received through a variety of inhibitory and activating receptors expressed on their cell surface. Narni-Mancinelli et al. (p. 344) now show that signaling through the activating receptor NKp46 paradoxically keeps NK cell responses in check. NK cells from mice with disrupted NKp46 expression were hyperresponsive to stimulation and better protected against viral infection. NK cell responses, which are part of the early response to infection, may thus need to be carefully tuned to ensure optimal initiation of adaptive immunity and formation of protective long-lived memory cells. The activating receptor NKp46 is important for keeping the responses of natural killer cells in check. Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46. The down-regulation of NK cell activity by NKp46 was associated with the silencing of the Helios transcription factor in NK cells. NKp46 was critical for the subsequent development of antiviral and antibacterial T cell responses, which suggests that the regulation of NK cell function by NKp46 allows for the optimal development of adaptive immune responses. NKp46 blockade enhanced NK cell reactivity in vivo, which could enable the design of immunostimulation strategies in humans.

Transduction of cytotoxic signals in natural killer cells: a general model of fine tuning between activatory and inhibitory pathways in lymphocytes

Summary: NK‐cells axe large granular lymphocytes, which are capable of exerting two major types of effector function, cell cytotoxicity and lymphokine secretion. NK ‐cells can exert cell cytotoxicity in one of two ways. First, NK‐cells are able to recognize and to induce the lysis of antibody‐coated target ceils during antibody‐dependent cell cytotoxicity (ADCC). Second, during natural cytotoxicity NK cells are also able to recoginze to induce the lysis of a variety of target cell, including primarily virus‐infected cells as well as tumor cells. Recently, a novel mechanism has been elucidated which controls NK‐cell‐activation programs and which is based on the cell surface expression of killer‐cell inhibitory receptors (KIR). We will review here the molecular dissection of this inhibitory signalling pathway which utilizes immunoreceptor tyrosine‐based inhibition motifs (ITIM) expressed in KIR intracytoplasmic domain. We will also show that this strategy used by NK‐cells to regulate their effector functions a general decision mechanism which exists not only in T‐ and B‐lymphocytes, but also in a variety of other hematopoietic cells.

Homophilic interaction of NTBA, a member of the CD2 molecular family: Induction of cytotoxicity and cytokine release in human NK cells

NK‐T‐B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA‐Fc*). NTBA‐Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA‐Fc* also binds to NTBA+ but not to NTBA– T cell lines. Enzyme‐linked immunosorbent assays, plasmon resonance analysis, as well as NTBA‐Fc*‐mediated down‐regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA‐Fc* promoted a strong production of IFN‐γ and TNF‐α. Moreover, NTBA‐transfected targets displayed increased susceptibility to NK‐mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti‐NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

Regulation of Inhibitory and Activating Killer-Cell Ig-Like Receptor Expression Occurs in T Cells After Termination of TCR Rearrangements

A small fraction of T cells expresses killer-cell Ig-like receptors (KIR), a family of MHC class I-specific receptors that can modulate TCR-dependent activation of effector functions. Although KIR+ cells are enriched within Ag-experienced T cell subsets, the precise relationships between KIR+ and KIR− T cells and the stage of KIR induction on these lymphocytes remain unclear. In this study, we compared KIR− and KIR+ αβ T cell clones, sorted by means of the CD158b (KIR2DL2/KIR2DL3/KIR2DS2) specific mAb GL183. We isolated several pairs of CD158b+ and CD158b− αβ T cell clones sharing identical productive and nonproductive TCR transcripts. We showed that expression of functional KIR on T cells is regulated after termination of TCR rearrangements. Transcriptional regulation of KIR genes was documented in multiple T cell clones generated from the same donor, and the presence of KIR transcripts was also detected in KIR− T cells. These results document a complex regulation of KIR expression in T cells at both pre and posttranscriptional levels, under the control of yet undefined signals provided in vivo.

Induction of B7-H6, a ligand for the natural killer cell–activating receptor NKp30, in inflammatory conditions

B7-H6, a member of the B7 family of immunoreceptors, is as a cell-surface ligand for the NKp30-activating receptor expressed on natural killer cells. B7-H6 is not detected in normal human tissues at steady state but is expressed on tumor cells. However, whether B7-H6 can be expressed in other conditions remains unknown. We analyzed here the pathways that lead to the expression of B7-H6 in nontransformed cells. In vitro, B7-H6 was induced at the surface of CD14(+)CD16(+) proinflammatory monocytes and neutrophils upon stimulation by ligands of Toll-like receptors or proinflammatory cytokines such as interleukin-1β and tumor necrosis factor α. In these conditions, a soluble form of B7-H6 (sB7-H6) was also produced by activated monocytes and neutrophils. In vivo, B7-H6 was expressed on circulating proinflammatory CD14(+)CD16(+) monocytes in a group of patients in sepsis conditions, and was linked to an increased mortality. sB7-H6 was selectively detected in the sera of patients with gram-negative sepsis and was associated with membrane vesicles that co-sedimented with the exosomal fraction. These findings reveal that B7-H6 is not only implicated in tumor immunosurveillance but also participates in the inflammatory response in infectious conditions.

CD146 and its soluble form regulate monocyte transendothelial migration.

Objectives—During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration. Among molecules at the interendothelial junction, CD146 is involved in cell–cell cohesion and permeability, but its role in monocyte transmigration is unknown. Methods and Results—TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes. Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. Conclusions—Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation.

Pathologies Associated with Serum IgG4 Elevation

Statement of Purpose. IgG4-related disease (IgG4-RD) is usually associated to an increase of serum IgG4 levels. However other conditions have also been associated to high serum IgG4 levels. Methods. All IgG subclasses analyses performed in our hospital over a one-year period were analyzed. When IgG4 level were over 1.35 g/L, the patients clinical observation was analyzed and both final diagnosis and reason leading to IgG subclasses analysis were recorded. Only polyclonal increases of IgG4 were considered. Summary of the Results. On 646 IgG subclass analysis performed, 59 patients had serum IgG4 over 1.35 g/L. The final diagnosis associated to serum IgG4 increase was very variable. Most patients (25%) presented with repeated infections, 13.5% with autoimmune diseases, and 10% with IgG4-RD. Other patients presented with cancer, primary immune deficiencies, idiopathic interstitial lung disease, cystic fibrosis, histiocytosis, or systemic vasculitis and 13.5% presented with various pathologies or no diagnosis. Mean IgG4 levels and IgG4/IgG ratio were higher in IgG4-RD than in other pathologies associated to elevated IgG4 levels. Conclusions. Our study confirms that elevation of serum IgG4 is not specific to IgG4-RD. Before retaining IgG4-RD diagnosis in cases of serum IgG4 above 1.35 g/L, several other pathological conditions should be excluded.

PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma

Programmed Death-1 (PD-1), an inhibitory receptor expressed by activated lymphocytes, is involved in regulating T- and B-cell responses. PD-1 and its ligands are exploited by a variety of cancers to facilitate tumor escape through PD-1-mediated functional exhaustion of effector T cells. Here, we report that PD-1 is upregulated on Natural Killer (NK) cells from patients with Kaposi sarcoma (KS). PD-1 was expressed in a sub-population of activated, mature CD56dimCD16pos NK cells with otherwise normal expression of NK surface receptors. PD-1pos NK cells from KS patients were hyporesponsive ex vivo following direct triggering of NKp30, NKp46 or CD16 activating receptors, or short stimulation with NK cell targets. PD-1pos NK cells failed to degranulate and release IFNγ, but exogenous IL-2 or IL-15 restored this defect. That PD-1 contributed to NK cell functional impairment and was not simply a marker of dysfunctional NK cells was confirmed in PD-1-transduced NKL cells. In vitro, PD-1 was induced at the surface of healthy control NK cells upon prolonged contact with cells expressing activating ligands, i.e. a condition mimicking persistent stimulation by tumor cells. Thus, PD-1 appears to plays a critical role in mediating NK cell exhaustion. The existence of this negative checkpoint fine-tuning NK activation highlights the possibility that manipulation of the PD-1 pathway may be a strategy for circumventing tumor escape not only from the T cell-, but also the NK-cell mediated immune surveillance.

Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia

CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases.