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. 2020 Aug 28;11:2028. doi: 10.3389/fimmu.2020.02028

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Copyright © 2020 Jamali, Hadjati, Madjd, Mirzaei, Thalheimer, Agarwal, Bonig, Ullrich and Hartmann.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Chemotaxis of NK cells is enhanced by CXCR4 overexpression. The migratory potential of genetically modified NK cells was evaluated in a migration assay using transwell plates. CAR NK cells were generated by transduction of primary NK cells with huCAR19-LV or huCAR19.CXCR4-LV particles. Untransduced primary NK cells cultured in parallel were used as a control. (A) Genetically modified or untransduced NK cells were placed in the upper chamber of the transwell plate and analyzed for their potential to migrate toward a lower chamber containing SDF-1 (CXCL12). The number of migrated NK cells was counted by flow cytometry. n = 4 with technical replicates. (B) Migration potential of NK cells was blocked by the CXCR4 antagonist AMD3100. Untreated or AMD3100 preincubated NK cells were placed in the upper chamber of the transwell plate and analyzed for their potential to migrate in the presence or absence of SDF-1. The number of migrated NK cells was counted by flow cytometry. The experiment was done in technical replicates. (C) A coculture assay was performed (in a 1:1 ratio) with migrated NK cells post chemotaxis to determine their cytotoxic potential. Specific target cell lysis was determined by decrease in the percentage of living CD19-positive cells analyzed by flow cytometry. n = 3. The experiment was done in technical replicates. Bar diagram show mean value, SD, and significance. **p < 0.005; ***p < 0.001; ns, non-significant by unpaired t test. (D) The migration potential of CSFE-labeled NK cells with or without AMD3100 pretreatment toward BMSCs plated in the lower wells. Representative fluorescent microscopic pictures of migrated, CFSE-labeled NK cells (right) as well as quantitative data by flow cytometry (left) is shown. Bar diagram shows individual results as well as mean values and standard deviation. The experiment was carried out once with purified NK cells from two different donors.