Examination of IgG Fc Receptor CD16A and CD64 Expression by Canine Leukocytes and Their ADCC Activity in Engineered NK Cells

Abstract

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.

Global and cell type-specific immunological hallmarks of severe dengue progression

“…Yet, their abundance is markedly decreased in the circulation, and they demonstrate increased exhaustion based on increased LAG3 expression in SDp, and PD-L1 expression detected by CyTOF 17 , suggesting reduced killing of DENV-infected cells. Alternatively, since CD16 and IFNγ- mediated CD64 expression on NK cells have been implicated in antibody-dependent cellular cytotoxicity (ADCC), one mechanism of infected-cell killing 35, 36, 37 , this expression pattern combined with the higher IFNγ secretion we detected in serum of SDp than D, suggest that ADCC may be induced in SDp.…”

Improved characterization and translation of NK cells for canine immunotherapy

“…At the time of this publication, the American Veterinary Medical Association (AVMA) Animal Health Studies Database lists five trials currently recruiting, and 35 trials with completed recruitment based on a search for “immunotherapy” in dogs with cancer. Studies from the University of Minnesota have demonstrated increased Antibody-Dependent Cellular Cytotoxicity (ADCC) efficacy in engineered human NK cells expressing recombinant CD64, opening the doors for the development of engineered canine NK cells that have similar effector capabilities ( 44 ). Investigators at The Ohio State University have simultaneously made progress in attempting to improve adoptive NK cell products for canine immunotherapy by imprinting NK cells with TGF-β during expansion to override potential inhibition in the TME ( 45 ).…”

Effects of faecal microbiota transplantation on the small intestinal mucosa in systemic sclerosis

“…With IHC analysis, we saw a significant reduction in podoplanin expression in lymphatic vessels and CD64 expression in the epithelium from weeks 0–2 in the duodenum of FMT-treated patients. CD64 is a high-affinity receptor for IgG (Fcγ-R1) and is constitutively expressed in the intestinal epithelium [ 23 , 24 ]. Furthermore, it has been implicated that in the context of intestinal mucosal inflammation, CD64 modulates the response against microbial challenges [ 25 ].…”