Engineering Receptor Expression on Natural Killer Cells Through Trogocytosis.

Trogocytosis is a rapid contact-dependent process by which lymphocytes acquire membrane patches from the target cells ('donor' cells) with which they interact and this phenomenon has been shown to occur in various immune cells. The surface molecules acquired through trogocytosis are functionally incorporated in the 'acceptor' cells transiently. We had previously demonstrated that trogocytosis can be utilized in place of gene transfer to engineer surface receptor expression on NK cells for adoptive immunotherapy applications. In this chapter, we describe detailed protocol for trogocytosis-co-culture of NK cell with the donor cell line, phenotypic assessment of receptor uptake and persistence, and assessment of NK cell function (migration) following receptor acquisition.

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

Engineering lymph node homing of ex vivo-expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7.

Natural killer (NK) cells have gained significant attention in adoptive immunotherapy for cancer. Consequently, novel methods of clinical-grade expansion of NK cells have emerged. Subsets of NK cells express a variety of chemokine receptors. However, to expand the scope of adoptively transferred NK cell homing to various malignancies, expression of corresponding chemokine receptors on NK cells is essential. Here, we have explored the use of trogocytosis as a tool to transiently express the chemokine receptor CCR7 on expanded human NK cells with the aim to enhance their homing to lymph nodes. We generated a K562-based "donor" cell line expressing CCR7, Clone9.CCR7, to transfer CCR7 onto NK cells via trogocytosis. CCR7 expression occurred in 80% of expanded NK cells within 1 hour after coculture with Clone9.CCR7. After removal of the donor cells from the coculture, the CCR7 expression on NK cells steadily declined to baseline levels by 72 hours. The acquired CCR7 receptors mediated in vitro migration of NK cells toward CCL19 and CCL21 and increased the lymph node homing by 144% in athymic nude mice. This is the first report on exploiting trogocytosis to rapidly and transiently modify lymphocytes, without direct genetic intervention, for adoptive transfer.