Stephan Meinke

Regulation of NK cell activity by 2B4, NTB-A and CRACC.

2B4, NTB-A and CRACC are members of the recently defined family of SLAM-related receptors. Here we review the role of these receptors for the regulation of Natural Killer cell function and describe the current knowledge about the signal transduction of these receptors. Finally, we critically analyze some controversial data about the function of 2B4 in mouse and man.

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‐α. 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.

Targets for Ibrutinib Beyond B Cell Malignancies

Ibrutinib (Imbruvica™) is an irreversible, potent inhibitor of Brutons tyrosine kinase (BTK). Over the last few years, ibrutinib has developed from a promising drug candidate to being approved by FDA for the treatment of three B cell malignancies, a truly remarkable feat. Few, if any medicines are monospecific and ibrutinib is no exception; already during ibrutinibs initial characterization, it was found that it could bind also to other kinases. In this review, we discuss the implications of such interactions, which go beyond the selective effect on BTK in B cell malignancies. In certain cases, the outcome of ibrutinib treatment likely results from the combined inhibition of BTK and other kinases, causing additive or synergistic, effects. Conversely, there are also examples when the clinical outcome seems unrelated to inhibition of BTK. Thus, more specifically, adverse effects such as enhanced bleeding or arrhythmias could potentially be explained by different interactions. We also predict that during long‐term treatment bone homoeostasis might be affected due to the inhibition of osteoclasts. Moreover, the binding of ibrutinib to molecular targets other than BTK or effects on cells other than B cell‐derived malignancies could be beneficial and result in new indications for clinical applications.

Random aggregates in newly produced platelet units are associated with platelet activation and release of the immunomodulatory factors sCD40L and RANTES

In connection with platelet (PLT) production, random but transient aggregates sometimes form in the newly produced units. The underlying mechanisms as well as the impact on cellular level of this phenomenon are unknown. Hypothetically, random occurrence of aggregates may induce biochemical changes leading to PLT activation and release of immunomodulatory factors from the PLTs.

NK cell cytotoxicity mediated by 2B4 and NTB-A is dependent on SAP acting downstream of receptor phosphorylation

2B4 (CD244) and NK-T-B-antigen (NTB-A, CD352) are activating receptors on human natural killer (NK) cells and belong to the family of signaling lymphocyte activation molecule (SLAM)-related receptors (SRR). Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SLAM-associated protein (SAP) and Ewings sarcoma-activated transcript-2 (EAT-2). X-linked lymphoproliferative syndrome (XLP) is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.

Platelets made HLA deficient by acid treatment aggregate normally and escape destruction by complement and phagocytes in the presence of HLA antibodies

The presence of antibodies against HLA Class I can lead to platelet (PLT) transfusion refractoriness, that is, the repeated failure to achieve adequate posttransfusion PLT count increments. PLT refractoriness can be overcome by transfusion of HLA‐matched donor PLTs. A different approach is to remove HLA from the PLT surface using low pH. Previous case studies using HLA‐stripped PLTs showed encouraging but inconsistent results and lacked information on the biologic effects of acid treatment on PLT function as well as sensitivity to PLT destruction in the presence of HLA antibodies.

Sensitive detection of platelet-specific antibodies with a modified MAIPA using biotinylated antibodies and streptavidin-coated beads.

We have developed a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) with enhanced sensitivity in detecting antibodies against human platelet antigens (HPA), using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry. The beads-MAIPA gave superior signal-to-noise resolution (>10-fold higher) for detection of anti-HPA-1a and anti-HPA-5b compared with the in-house standard MAIPA. Also, efficient and reproducible detection of anti-HPA-15 (CD109) was shown. The enhanced sensitivity was confirmed using WHO International Reference Reagents for anti-HPA-1a, anti-HPA-3a and anti-HPA-5b, which allowed comparison of detection endpoints with other laboratories. Finally, the beads-MAIPA was validated for quantification of anti-HPA-1a. The lower limit of quantification was 0.4IU/mL for beads-MAIPA, compared to 1IU/mL previously reported for standard MAIPA. Based on improved performance against all HPA-antibodies tested, the beads-MAIPA has replaced the standard MAIPA in our laboratory in diagnostics of conditions due to HPA-immunization, such as fetal and neonatal alloimmune thrombocytopenia (FNAIT).

Characterisation of maternal human leukocyte antigen class I antibodies in suspected foetal and neonatal alloimmune thrombocytopenia

To investigate the specificities and level of HLA class I antibodies in selected cases referred for suspected foetal and neonatal alloimmune thrombocytopenia (FNAIT).

The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl‐1 (c‐Abl)‐mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c‐Cbl, suggesting that NK cell education might involve c‐Abl. Mice deficient in c‐Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c‐Abl‐deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC‐1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN‐γ after activating receptor stimulation. Consistent with these results, c‐Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c‐Abl and that c‐Abl plays no major role in NK cell education in the mouse.

Independent control of natural killer cell responsiveness and homeostasis at steady-state by CD11c+ dendritic cells

During infection and inflammation, dendritic cells (DC) provide priming signals for natural killer (NK) cells via mechanisms distinct from their antigen processing and presentation functions. The influence of DC on resting NK cells, i.e. at steady-state, is less well studied. We here demonstrate that as early as 1 day after DC depletion, NK cells in naïve mice downregulated the NKG2D receptor and showed decreased constitutive phosphorylation of AKT and mTOR. Subsequently, apoptotic NK cells appeared in the spleen concomitant with reduced NK cell numbers. At 4 days after the onset of DC depletion, increased NK cell proliferation was seen in the spleen resulting in an accumulation of Ly49 receptor-negative NK cells. In parallel, NK cell responsiveness to ITAM-mediated triggering and cytokine stimulation dropped across maturation stages, suggestive of a functional deficiency independent from the homeostatic effect. A role for IL-15 in maintaining NK cell function was supported by a gene signature analysis of NK cell from DC-depleted mice as well as by in vivo DC transfer experiments. We propose that DC, by means of IL-15 transpresentation, are required to maintain not only homeostasis, but also function, at steady-state. These processes appear to be regulated independently from each other.