The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.

NKp44 (NCR2) is a distinct member of Natural Cytotoxicity Receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in –cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in –trans and therefore enhances NKp44 activation potential and NK cell functional response.

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Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors’ sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.

The understanding of the activation mechanism of natural killer (NK) cells has been traditionally based on the biochemical interaction of NK cell receptors with the ligands presenting on target cells. Yet, how physical features of the NK cell environment, such as stiffness and nanoscale topography, regulate the activity of NK cells, is unknown. An artificial microenvironment is developed for NK cell stimulation based on variably elongated nanowires functionalized with activating antigens and used it as a tunable model system to study the separate and cumulative effects of elasticity and nanotopography on NK cell activation. It is found that late signaling, degranulation, and cytokine production in NK cells consistently vary with nanowire length, and it is maximized for the longest nanowires, measuring ≈20 µm in length. Intriguingly, a similar trend is observed for nanowires lacking antigens, albeit with a lower activation magnitude overall, indicating that chemical and mechanical stimuli independently determine the activation of NK cells. The demonstrated tuning of the activation of NK cells by nanoscale physical features of their environment not only provides an important insight into the regulation mechanism of these lymphocytes, but also paves the way to rationally designed nanomaterials for controlled ex vivo activation of NK cells in immunotherapy.

The role of juxtaposition of activating and inhibitory receptors in signal inhibition of cytotoxic lymphocytes remains strongly debated. The challenge lies in the lack of tools that allow simultaneous spatial manipulation of signaling molecules. To circumvent this, we produced a nanoengineered multifunctional platform with molecular-scale spatial control of ligands, which was applied to elucidate KIR2DL1-mediated inhibition of NKG2D signaling—receptors of natural killer cells. This platform was conceived by bimetallic nanodot patterning with molecular-scale registry, followed by a ternary functionalization with distinct moieties. We found that a 40-nm gap between activating and inhibitory ligands provided optimal inhibitory conditions. Supported by theoretical modeling, we interpret these findings as a consequence of the size mismatch and conformational flexibility of ligands in their spatial interaction. This highly versatile approach provides an important insight into the spatial mechanism of inhibitory immune checkpoints, which is essential for the rational design of future immunotherapies.

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.

IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: “S2A”. Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.