Anne Marie-Cardine

Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity

Circulating human natural killer (NK) lymphocytes have been functionally defined by their ability to exert cytotoxic activity against MHC class I-negative target cell lines, including K562. Therefore, it was proposed that NK cells recognized the “missing self.” We show here that the Ig-like CD160 receptor expressed by circulating CD56dim+ NK cells or IL-2-deprived NK cell lines is mainly involved in their cytotoxic activity against K562 target cells. Further, we report that HLA-C molecules that are constitutively expressed by K562 trigger NK cell lysis through CD160 receptor engagement. In addition, we demonstrate, with recombinant soluble HLA-Cw3 and CD160 proteins, direct interaction of these molecules. We also find that CD158b inhibitory receptors partially interfere with CD160-mediated cytotoxicity, whereas CD94/CD159a and CD85j have no effect on engagement with their respective ligands. Thus, CD160/HLA-C interaction constitutes a unique pathway to trigger NK cell cytotoxic activity.

Semaphorin CD100 from Activated T Lymphocytes Induces Process Extension Collapse in Oligodendrocytes and Death of Immature Neural Cells

An inappropriate cross talk between activated T lymphocytes infiltrating the CNS and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious cross talk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific Abs of soluble CD100 (sCD100)/semaphorin 4D released by activated T cells, or treatment with rsCD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through receptors of the plexin family. The specific presence of sCD100 in the cerebrospinal fluid and of CD100-expressing T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed to the potential pathological effect of sCD100 in the CNS. Thus, our results show that CD100 is a new important element in the deleterious T cell-neural cell cross talk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and human T lymphotropic virus type 1-associated myelopathy.

Cutting Edge: Engagement of CD160 by its HLA-C Physiological Ligand Triggers a Unique Cytokine Profile Secretion in the Cytotoxic Peripheral Blood NK Cell Subset

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-γ, TNF-α, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.

SC5 mAb Represents a Unique Tool for the Detection of Extracellular Vimentin as a Specific Marker of Sézary Cells

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4+CD45RO+ T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.

A Soluble Form of the MHC Class I-Specific CD160 Receptor Is Released from Human Activated NK Lymphocytes and Inhibits Cell-Mediated Cytotoxicity

CD160 is a GPI-anchored lymphocyte surface receptor in which expression is mostly restricted to the highly cytotoxic CD56dimCD16+ peripheral blood NK subset. We previously reported that MHC class I (MHC-I) molecules bind to CD160 receptors on circulating NK lymphocytes and that this interaction triggers their cytotoxic activity and cytokine production. We also observed that CD160 surface expression on NK cells is down-modulated upon activation with PMA or IL-2. In this study, we further report that short-time incubation of NK lymphocytes with IL-15 converts the membrane-bound CD160 to a soluble form through a proteolytic cleavage involving a metalloprotease. Thus, CD160 is no longer detected at the cell surface, but can be immunoprecipitated from the NK cell culture medium. Interestingly, CD160 transcript remains highly expressed during the process of protein shedding. In addition, we demonstrate that CD160 mRNA synthesis can be induced in CD56bright separated lymphocytes following exposure to IL-15. By producing a Flag-tagged soluble CD160 protein, we establish that its binding to MHC-I molecules results in the inhibition of the cytotoxic CD8+ T lymphocyte activity and of the CD160-mediated NK cell cytotoxicity. Thus, we show that activated NK lymphocytes release a soluble form of CD160 that functionally impairs the MHC-I-specific cytotoxic CD8+ T lymphocyte responsiveness.

Absolute CD3+ CD158k+ lymphocyte count is reliable and more sensitive than cytomorphology to evaluate blood tumour burden in Sézary syndrome

Background  CD158k/KIR3DL2 is a specific marker for Sézary cells which can be used to diagnose Sézary syndrome (SS) in erythrodermic patients with abnormal circulating T cells.

Structural and functional dissection of the cytoplasmic domain of the transmembrane adaptor protein SIT (SHP2-interacting transmembrane adaptor protein)

SIT (SHP2‐interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR‐mediated recruitment of SH2 domain‐containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2‐containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine‐based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR‐mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2‐binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine‐based signaling motif Y168 ASV completely abrogates the ability of SIT to inhibit T cell activation. Co‐precipitation experiments revealed that the tyrosine kinase p50csk could represent the negative regulatory effector molecule that binds to this motif.

Identification and Characterization of a Transmembrane Isoform of CD160 (CD160-TM), a Unique Activating Receptor Selectively Expressed upon Human NK Cell Activation

CD160 has been initially identified as a GPI-anchored MHC-class I activating receptor mainly expressed on peripheral blood NK cells. Herein, we report the identification of three additional CD160-related mRNAs generated through alternative splicings of the CD160 gene, among which one encoded a putative CD160 transmembrane isoform (CD160-TM). We first establish that CD160-TM surface expression is highly restricted to NK cells and is activation-dependent. Additionally, we provide evidence that CD160-TM represents a novel activating receptor, as assessed by the increased CD107a NK cell surface mobilization observed upon its engagement. Finally, we demonstrate that the CD160-TM cytoplasmic tail is by itself sufficient to mediate the recruitment of Erk1/2 signaling pathway, and that the initiation of this activation process is dependent on the Src-family kinase p56lck. The identification of CD160-TM therefore provides new possibilities regarding the role of CD160 isoforms in the regulation of NK cell functions.

Expression and Function of the Natural Cytotoxicity Receptor NKp46 on Circulating Malignant CD4+ T Lymphocytes of Sézary Syndrome Patients

The natural cytotoxicity receptors NKp30, NKp44, and NKp46 were identified as activating receptors mainly expressed by natural killer (NK) lymphocytes. In this study we show that peripheral blood malignant CD4(+) T lymphocytes from patients with Sézary syndrome, an aggressive form of cutaneous T-cell lymphoma, express NKp46 at their cell surface. Although NKp46 does not behave as an independent functional receptor, its engagement provides a strong inhibiting signal on the malignant T lymphocyte CD3-induced proliferation. We show that this inhibition is correlated with a decreased phosphorylation of the CD3ζ chain associated with NKp46 and/or the TCR/CD3 complexes. Our results reveal that in addition to KIR3DL2/CD158k expression, NKp46 could represent an additional marker on the circulating malignant T lymphocytes of Sézary patients, where it displays an as yet unreported function of inhibitory co-receptor able to interfere with the processes governing their CD3-dependent proliferation.

Induction of hyperphosphorylation and activation of the p56lck protein tyrosine kinase by phenylarsine oxide, a phosphotyrosine phosphatase inhibitor

The T cell protein tyrosine kinase p56lck is implicated in thymic development and mitogenic activation of T lymphocytes, and is itself regulated by reversible tyrosine phosphorylation. When phenylarsine oxide (PAO), a membrane-permeable inhibitor of phosphotyrosine phosphatases, was added to Jurkat T leukemia or LSTRA thymoma cells, the phosphate content of p56lck increased rapidly. The sites of increased phosphorylation were mapped to Tyr-192, Tyr-394 and Tyr-505. Hyperphosphorylated p56lck displayed retarded mobility on SDS gels, unaltered or marginally increased cytoskeletal association, and its catalytic activity changed in a biphasic manner; during the first 10-20 min of PAO-treatment the activity increased and then it declined to very low values within 1-2 hr. Our data suggest that p56lck contains both positive and negative regulatory sites which are constantly dephosphorylated at an unexpectedly high rate by cellular phosphotyrosine phosphatases.