Dataset Information

Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

ABSTRACT: Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by FcγRIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

SUBMITTER: Ross P

PROVIDER: S-EPMC11326286 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

Ross Patrick P Fatima Hijab H Leaman Dan P DP Matthias Jessica J Spencer Kathryn K Zwick Michael B MB Henderson Scott C SC Mace Emily M EM Murin Charles Daniel CD

bioRxiv : the preprint server for biology 20240810

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc<i>γ</i>RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we ...[more]

PMID: 39149244

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Project description:Human natural killer (NK) cells can target tumor cells in an antigen-specific manner by the recognition of cell bound antibodies. This process induces antibody-dependent cell-mediated cytotoxicity (ADCC) and is exclusively mediated by the low affinity IgG Fc receptor CD16A (FcγRIIIA). Exploiting ADCC by NK cells is a major area of emphasis for advancing cancer immunotherapies. CD64 (FcγRI) is the only high affinity IgG FcR and it binds to the same IgG isotypes as CD16A, but it is not expressed by human NK cells. We have generated engineered human NK cells expressing recombinant CD64 with the goal of increasing their ADCC potency. Preclinical testing of this approach is essential for establishing efficacy and safety of the engineered NK cells. The dog provides particular advantages as a model, which includes spontaneous development of cancer in the setting of an intact and outbred immune system. To advance this immunotherapy model, we cloned canine CD16A and CD64 and generated specific mAbs. We report here for the first time the expression patterns of these FcγRs on dog peripheral blood leukocytes. CD64 was expressed by neutrophils and monocytes, but not lymphocytes, while canine CD16A was expressed at high levels by a subset of monocytes and lymphocytes. These expression patterns are similar to that of human leukocytes. Based on phenotypic characteristics, the CD16A+ lymphocytes consisted of T cells (CD3+ CD8+ CD5dim α/β TCR+) and NK cells (CD3- CD5- CD94+), but not B cells. Interestingly, the majority of canine CD16A+ lymphocytes were from the T cell population. Like human CD16A, canine CD16A was downregulated by a disintegrin and metalloproteinase 17 (ADAM17) upon leukocyte activation, revealing a conserved means of regulation. We also directly demonstrate that both canine CD16A and CD64 can induce ADCC when expressed in the NK cell line NK-92. These findings pave the way to engineering canine NK cells or T cells with high affinity recombinant canine CD64 to maximize ADCC and to test their safety and efficacy to benefit both humans and dogs.

Project description:Follicular lymphoma (FL) is the second most frequent subtype of B non-Hodgkin's lymphomas (NHL) for which the treatment is based on the use of anti-CD20 mAbs. NK cells play a crucial role in their mechanism of action and the number of these cells mediating antibody-dependent cell cycotoxicity (ADCC) in the peripheral blood of FL patients predict the outcome. However, their presence in FL biopsies, their activation and their role have been poorly investigated. Moreover, in vitro studies have not deciphered the exact signaling cascades triggered by NK cells in presence of anti-CD20 mAbs on both effector and target cells in a relevant FL model. We performed in silico analyses and ex vivo functional assays to determine the presence and the activation status of NK cells in FL biopsies. We modelized ADCC phenomenon by developing a co-culture model composed by 3D-cultured FL cells and NK cells. Thus, we investigated the biological effect of anti-CD20 mAbs by fluorescent microscopy and the phosphorylation status of survival pathways by cell bar coding phosphoflow in target cells. In parallel, we measured the status of activation of downstream FcγRIIIa signaling pathways in effector cells and their activation (CD69, perforin, granzyme B, IFNγ) by flow cytometry. We determined by in vivo experiments the effects of anti-CD20 mAbs in presence of NK cells in SCID-Beige engrafted FL mice. Here, we show that functional NK cells infiltrate FL biopsies, and that their presence tends to correlate with the survival of FL patients. Using our 3D co-culture model, we show that rituximab and GA101 are able to promote degranulation, CD69 expression, IFNγ production and activate FcγRIIIa signaling cascade in NK cells, and inhibit survival pathways and induce apoptosis in FL cells. The effect of GA101 seems to be more pronounced as observed in vivo in a xenograft FL model. This study strongly supports the role of NK cells in FL and highlights the application of the 3D co-culture model for in vitro validation.

Dataset Information

Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

Publications

Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

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