Figure 1.
Immunophenotypic features of expanded NK cells (acceptor cells) and K562-antiCD19BBζ cells (feeder cells).
A. Expression of CD56 and CD3 on peripheral blood mononuclear cells from a healthy donor was examined after 1 week (top row) of co-culture with irradiated (IR, left column) or freeze/thaw-treated (F, right column) K562-mb15-41BBL cells at a low dose (10 U/mL) of IL-2. The T cells were removed using CD3 Dynabeads, generating cell populations comprising >95% CD56+CD3- NK cells (bottom row). B. Percentage of CD56-positive cells within NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells prior to and after CD3 depletion on day 7. The data are presented as the mean of values obtained using 3 unrelated NK donors. Error bars represent the SD. C. Histogram illustrating the anti-CD19 expression on K562 cells (control, shaded histogram) and K562-antiCD19BBζ cells (feeder cells, open histogram).
Figure 2.
Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis.
A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p<0.05; two-tailed paired Student's t-tests).
Figure 3.
Immunofluorescence analysis for trogocytosis.
A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure 4.
Degranulation and cytotoxicity assays.
A. Incubation of NK cells with RS4;11 cells induced degranulation, which was significantly higher in NK cells co-cultured with K562-antiCD19BBζ cells than in NK cells cultured with control K562 cells. Percentages of CD56-positive cells from 3 donors expressing CD107a after a 4-h RS4;11 stimulation are shown. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. B. Flow cytometric dot plots illustrating the assay used to measure cell killing. Results for RS4;11 and OP-1 cell lines are shown. Tumor cells were either cultured alone (left panel), with NK cells previously co-cultured with K562 cells (middle panel), or with NK cells previously co-cultured with K562-antiCD19BBζ cells (right panel). Residual viable target cells, which were defined as calcein AM-positive and propidium iodide (PI)-negative, are shown in the bottom right corner of each panel, and the percentages of viable cells are shown. C. Cytotoxicity of the non-B leukemia cell line (K562) and B-ALL cell lines (RS4;11, OP1, and SUP-B15) after 4-h co-culture with NK cells previously co-cultured with K562 cells (white circles) and K562-antiCD19BBζ cells (black circles) at the indicated E:T ratios. The equation [(tumor co-culture with NK cells)/(tumor alone)]×100%, represents the quantitative percentage of viable tumor cells. The cytotoxicity was calculated according the following equation: 100% – quantitative percentage of viable tumor cells. Each symbol corresponds to the mean of three values. *: significant increase compared with control cells (p<0.05; two-tailed paired Student's t-tests).
Figure 5.
NK cells that acquired anti-CD19 CARs were more cytotoxic against patient-derived primary B-ALL cells.
Cytotoxicity against B-ALL cells after 4-h co-culture with NK cells previously co-cultured with K562 cells (white circles) and K562-antiCD19BBζ cells (black circles) at the indicated E:T ratios. Each symbol corresponds to the mean of three values.