Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells

Mark, Christoph; Czerwinski, Tina; Roessner, Susanne; Mainka, Astrid; F, Horsch; Heublein, Lucas; Winterl, Alexander; Sanokowski, Sebastian; Richter, Sebastian; Bauer, Nina; Angelini, Thomas E.; Schuler, Gerold; Fabry, Ben; Voskens, Caroline J.

doi:10.1038/s41467-020-19094-0

Cited by 57 publications

(54 citation statements)

References 36 publications

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“…32,34,35 Interestingly, the loss of CD16 expression on NK cells by cryopreservation/thawing of human PBMCs has been reported in previous studies. 14,34,36 In the current study, the uCD56 dim NK-cell population was prominently observed in cryopreserved PBMCs from patients with COVID-19, but not healthy donors, indicating that NK cells become abnormally labile to ex vivo cellular stress, such as cryopreservation/thawing, during COVID-19. In the present study, we also showed that the frequency of CD62L 1 cells was significantly lower among uCD56 dim NK cells than among cCD56 dim NK cells (Fig E10 ), indicating that the ADAM17 activity is increased in uCD56 dim NK cells.…”

Section: Discussionmentioning

confidence: 50%

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients

Leem

Cheon

Lee

et al. 2021

Journal of Allergy and Clinical Immunology

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Background Our understanding of adaptive immune responses in COVID-19 patients is rapidly evolving, but information on the innate immune responses by natural killer (NK) cells is still insufficient. Objective We aimed to examine the phenotypic and functional status of NK cells and their changes during the course of mild and severe COVID-19. Methods We performed RNA sequencing (RNA-seq) and flow cytometric analysis of NK cells from patients with mild and severe COVID-19 at multiple time points in the course of the disease using cryopreserved peripheral blood mononuclear cells (PBMCs). Results In RNA-seq analysis, the NK cells exhibited distinctive features compared to healthy donors, with significant enrichment of pro-inflammatory cytokine-mediated signaling pathways. Intriguingly, we found that the unconventional CD56 dim CD16 neg (uCD56 dim ) NK cell population expanded in cryopreserved PBMCs from COVID-19 patients regardless of disease severity, accompanied by decreased NK cell cytotoxicity. The NK cell population was rapidly normalized alongside the disappearance of uCD56 dim NK cells and the recovery of NK cell cytotoxicity in mild COVID-19 patients, but this occurred slowly in severe COVID-19 patients. Conclusion The current longitudinal study provides a deep understanding of the NK cell biology in COVID-19.

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Section: Discussionmentioning

confidence: 50%

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients

Leem

Cheon

Lee

et al. 2021

Journal of Allergy and Clinical Immunology

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“…Our ultimate goal is to deploy this manufacturing pipeline for clinical use, requiring cryopreservation and banking of manufactured CAR-NK cells. Recent studies have shown that while cryopreservation may have little effect on NK viability, it can cause a loss of cytotoxic function 55 . Thus, we tested cytotoxicity of CAR-NK cells immediately after thaw or after overnight culture in media containing 100 IU/mL IL2 (Figure 3D).…”

Section: Resultsmentioning

confidence: 99%

“…CAR-NK cells showed high levels of degranulation, inflammatory cytokine production, and target cell killing compared to CAR-negative controls (Figure 3A – 3D). Interestingly, it has been shown that cryopreservation may lead to reduced cytotoxicity of NK cells 55 . An off-the-shelf product requires cryopreservation and banking of engineered cells.…”

Section: Discussionmentioning

confidence: 99%

Non-Viral Engineering of CAR-NK and CAR-T cells using theTc BusterTransposon System™

Pomeroy

Lahr

Chang

et al. 2021

Preprint

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Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome. Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function. Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

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“…Our experiments are based on natural killer (NK) cells from human donors, which are in vitro activated and expanded 16 . A number of 300.000 NK immune cells and 120.000 K562 tumor cells are mixed with ice-cold 1500 µ l acid-dissolved collagen solution (1.2 mg/ml) and pipetted in each well of a tissue-culture treated 6-well plate (Corning).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, cells are segmented from the background and classified with a convolutional neural network, using the same method as described in 16 . The network has been trained on 6 manually labeled minimum/maximum intensity projection images of NK cells, mixed with K562 cells.…”

Section: Methodsmentioning

confidence: 99%

Detecting long-range interactions between migrating cells

Metzner

Mark

et al. 2021

Sci Rep

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Chemotaxis enables cells to systematically approach distant targets that emit a diffusible guiding substance. However, the visual observation of an encounter between a cell and a target does not necessarily indicate the presence of a chemotactic approach mechanism, as even a blindly migrating cell can come across a target by chance. To distinguish between the chemotactic approach and blind migration, we present an objective method that is based on the analysis of time-lapse recorded cell migration trajectories: For each movement step of a cell relative to the position of a potential target, we compute a p value that quantifies the likelihood of the movement direction under the null-hypothesis of blind migration. The resulting distribution of p values, pooled over all recorded cell trajectories, is then compared to an ensemble of reference distributions in which the positions of targets are randomized. First, we validate our method with simulated data, demonstrating that it reliably detects the presence or absence of remote cell-cell interactions. In a second step, we apply the method to data from three-dimensional collagen gels, interspersed with highly migratory natural killer (NK) cells that were derived from two different human donors. We find for one of the donors an attractive interaction between the NK cells, pointing to a cooperative behavior of these immune cells. When adding nearly stationary K562 tumor cells to the system, we find a repulsive interaction between K562 and NK cells for one of the donors. By contrast, we find attractive interactions between NK cells and an IL-15-secreting variant of K562 tumor cells. We therefore speculate that NK cells find wild-type tumor cells only by chance, but are programmed to leave a target quickly after a close encounter. We provide a freely available Python implementation of our p value method that can serve as a general tool for detecting long-range interactions in collective systems of self-driven agents.

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Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells

Cited by 57 publications

References 36 publications

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients

Non-Viral Engineering of CAR-NK and CAR-T cells using theTc BusterTransposon System™

Detecting long-range interactions between migrating cells

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