Fig 2: Cryopreserved NK cells exert antitumor effect in an in vivo PDX mouse model. Ascites were collected from an NSG mouse that had PDX tumor injected intraperitoneally. Spheroids were obtained from the ascites, and 20,000 spheroids were injected in suspension back to the intraperitoneal (i.p.) cavity of female NSG mice and allowed to seed for 3 days. Mice were then treated with 2 doses of cryopreserved NK cells (1 × 107/dose, days 0 and 5) from two donors, which were thawed and directly injected i.p. along with IL-2 (25,000 U, 3×/week). Health was monitored periodically, and abdominal wash was collected from one mouse treated with frozen NK cells euthanized 64 days posttreatment. A schematic of the experiment is shown (A). Mice injected with frozen NK cells from donor 1 (blue dashed line) and donor 2 (red dotted line) had a significant increase in survival compared to untreated mice (black solid line) (B). Flow cytometry dot plots are shown for gating on CD45+ cells in the abdominal wash of a treated mouse euthanized on day 64 posttreatment (C). The percentages of hNKT (CD3+, CD56+), hNK (CD3-,CD56+), and hT cells (CD3+) were determined in the hCD45+ population. Statistical significance was determined by comparing survival curves using the Gehan–Breslow–Wilcoxon test.

Fig 3: Spatial distribution of dNK subsets at the feto-maternal interface at early pregnancyA. A representative immunohistochemistry image for CK7 (brown), labeling trophoblasts and uterine gland epithelium in a paraffin section of the feto-maternal interface at gestational week 7. Spongiosa, decidual spongiosa; Compacta, decidual compacta. B. Representative immunofluorescent images of sections adjacent to the section in panel A, showing the distribution of dNK1 cells (CD56+CD39+; white arrows) at the feto-maternal interface. C. Representative immunofluorescent images of sections adjacent to the section in panel A, showing the distribution of dNK3 cells (CD56+CD103+; white arrows) at the feto-maternal interface. In both panels, boxed areas from different regions of the feto-maternal interface on the left (scale bar, 100 µm) are zoomed in and shown in the middle (scale bar, 25 µm), and boxed areas in the middle are further zoomed in and shown on the right (scale bar, 10 µm). D. Representative immunofluorescent images showing the distribution of dNK4 cells (CD16+ PLAC8+; white arrows) in decidual compacta from Ctrl (top) and RM (bottom) pregnancies. Dotted lines indicate the position of uterine blood vessels. Scale bar, 25 µm.

Fig 4: Alterations of dNK subsets in the decidua of RM pregnancyA. Representative flow cytometry plots showing the strategy to sort dNK cells in human decidual tissues at gestational weeks 6–8, including dNK1 (CD3-CD56+CD39+), dNK3 (CD3-CD56+CD39-CD103+), and dNK4 (CD3-CD56+CD16+). B. Quantification of cell population of dNK1 and dNK3 (left panel), as well as dNK4 (right panel) subsets in decidual tissues from Ctrl (n = 5) and RM patients (n = 5 in left panel, and n = 11 in right panel). Significant difference between the two groups is analyzed using unpaired t-test. *, P < 0.05; **, P < 0.01. C. Violin plots illustrating the expression of typical differential genes (absolute log2 FC > 0.5 and Bonferroni-adjusted P < 0.05) in dNK1, dNK3, and dNK4 cells from Ctrl and RM patients. The differential expression analysis is performed using the method of non-parametric two-sided Wilcoxon rank sum test in Seurat.

Fig 5: NK cells are viable after cryopreservation. NK cells were expanded with PM21 particles (PM21-NK cells) from T-cell-depleted PBMCs obtained from multiple donors (N = 19). Cells were cryopreserved and stored in liquid nitrogen until use. Frozen PM21-NK cells were thawed and counted for number of viable NK cells, and no loss of NK cells was seen. Representative flow cytometry dot plots gating for viable (Draq7-) NK (CD3-, CD56+) cells immediately post-thaw are shown (A). Thawed NK cells were placed in media at 1 × 107 of viable cells and rested overnight at 37°C. After 16 h, the total number of viable NK cells was counted. Percent recovery at 16 h post-thaw was calculated by dividing the total number of viable cells, remaining after 16 h rest, by 1 × 107. Mean recovery was 73% ± 22% (B). Data are represented as a scatter plot with an error bar representing standard deviation.