2B4 engagement mediates rapid LFA-1 and actin-dependent NK cell adhesion to tumor cells as measured by single cell force spectroscopy.

Adhesion to tumor target cells is essential for initiation and execution of cellular cytotoxicity. In this study, we use single cell force spectroscopy to determine the exact biophysical values of the interaction forces between NK cells and tumor cells. We show that engagement of the activating NK cell receptor 2B4 can rapidly mediate an increase in the force necessary to separate NK cells from tumor cells, starting from 1 nN and increasing to 3 nN after only 120 s tumor cell contact. This early adhesion was mediated by the integrin LFA-1 and dependent on the actin cytoskeleton. The ability of NK cells to rapidly adhere to tumor target cells is consistent with their function in innate immune responses. Our data further suggest that a killing decision is already made within 120- 300 s of tumor cell contact, supporting the essential function of cell adhesion during the early phase of cellular cytotoxicity.

Endothelial cell ephrinB2-dependent activation of monocytes in arteriosclerosis.

OBJECTIVE: The expression of ephrinB2 in endothelial cells delineates their arterial phenotype and is a prerequisite for the development of the embryonic vasculature. Whereas the role of ephrinB2 in the microcirculation has been studied extensively, its expression and function in adult arteries is hardly understood. METHODS AND RESULTS: Our analyses showed that in mouse arteries, ephrinB2 is located on the luminal surface of endothelial cells and may physically interact with monocyte EphB receptors. Moreover, transdifferentiation of human monocytes into macrophages was associated with an increase in EphB2 expression, and exposing monocytes to immobilized ephrinB2 resulted in phosphorylation of the receptor followed by an increased expression of proinflammatory chemokines such as interleukin-8 and monocyte chemotactic protein-1/CCL2. Relating to the (patho)physiological relevance of these findings, immunofluorescence analyses revealed that ephrinB2 is most abundantly expressed in endothelial cells at arteriosclerosis predilection sites of the mouse aorta. Subsequent analyses indicated that monocyte adhesion to aortic segments abundantly expressing ephrinB2 is strongly enhanced and that endothelial cell ephrinB2 forward signaling is sufficient to upregulate cytokine expression in monocytes. CONCLUSIONS: These observations suggest a hitherto unknown link between vascular ephrinB2 expression and the proinflammatory activation of monocytes that may contribute to the pathogenesis of arteriosclerosis.

Functional analysis of bispecific antibody (EpCAMxCD3)-mediated T-lymphocyte and cancer cell interaction by single-cell force spectroscopy.

The atomic force microscopy (AFM) is a powerful tool to analyze forces generated on cellular interactions on the single-cell level. This highly sensitive device can record changes in force in the pico-Newton range, which equals single molecule bonds. Here, we have used single-cell force spectroscopy by AFM to investigate the interaction between T cells and tumor cells that is induced by the bispecific antibody HEA125xOKT3 (specificity anti-EpCAMxCD3). We show that HEA125xOKT3 induces a specific increase in adhesion force between T cells and cancer cells. The adhesive force that is generated on cell-cell contact is dependent on the applied force on initial contact and the duration of this initial contact. In summary, HEA125xOKT3 has been found to mediate contact formation by distinct processes. It induces direct cell-cell interaction, which results in the activation of T-cell signaling, facilitates the formation of supramolecular activation clusters and ultimately of an immune synapse.

Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool.

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.

Expression analysis of the ligands for the Natural Killer cell receptors NKp30 and NKp44.

BACKGROUND: The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer. METHODOLOGY/PRINCIPAL FINDINGS: Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G(2)/M phase. CONCLUSION/SIGNIFICANCE: These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands.

Identification of CLEC12B, an inhibitory receptor on myeloid cells.

Activation of immune cells has to be tightly controlled to prevent detrimental hyperactivation. In this regulatory process molecules of the C-type lectin-like family play a central role. Here we describe a new member of this family, CLEC12B. The extracellular domain of CLEC12B shows considerable homology to the activating natural killer cell receptor NKG2D, but unlike NKG2D, CLEC12B contains an immunoreceptor tyrosine-based inhibition motif in its intracellular domain. Despite the homology, CLEC12B does not appear to bind NKG2D ligands and therefore does not represent the inhibitory counterpart of NKG2D. However, CLEC12B has the ability to counteract NKG2D-mediated signaling, and we show that this function is dependent on the immunoreceptor tyrosine-based inhibition motif and the recruitment of the phosphatases SHP-1 and SHP-2. Using monoclonal anti-CLEC12B antibodies we found de novo expression of this receptor on in vitro generated human macrophages and on the human myelo-monocytic cell line U937 upon phorbol 12-myristate 13-acetate treatment, suggesting that this receptor plays a role in myeloid cell function.

Modulation of 2B4 (CD244) activity and regulated SAP expression in human NK cells.

The adapter protein SAP is important for the signal transduction of the family of SLAM-related receptors (SRR), which have important immune-modulating functions. The importance of SAP and SRR for a functional immune reaction becomes obvious in patients suffering from X-linked lymphoproliferative disease, which is characterized by non-functional SAP. Here we investigate the regulation of SAP expression in human NK cells. We demonstrate that SAP mRNA expression and protein levels are low in freshly isolated resting NK cells. IL-2 stimulation leads to an up-regulation of SAP expression, which can be enhanced by IL-12, the stimulation of TLR3 by polyinosinic-polycytidylic acid (poly(I:C))and to a lesser extent by IFN-alpha. EAT-2, a SAP-related adapter protein, is already detectable in resting NK cells and does not change its expression after IL-2 stimulation. The regulation of SAP has functional consequences for the stimulation of NK cell cytotoxicity by 2B4. In resting NK cells, 2B4 stimulation can only enhance NK cell lysis when co-triggered with other activating NK cell receptors. In IL-2-activated NK cells with high SAP expression the triggering of 2B4 alone is sufficient to induce NK cell cytotoxicity, demonstrating a correlation between the regulated SAP expression and the function of 2B4.