Article Figures & Data
Figures
- Figure 1.
Optimization of primary NK-cell redirection by CAR. A, Histogram showing CAR (empty) expression against background in mock (filled) transfected NK cells after various durations of cytokine (IL15) stimulation. B, Representative dot-plot overlays illustrating CAR expression (red) against background from mock (blue)-transfected NK cells over the next 4 days after transfection and different generations of division in culture. C, Summary graphs from two experiments with two donors included in each experiment (n = 4) illustrating CAR expression over 4 days after transfection and various generations of division. D, CAR constructs used in the study. E, Representative histograms showing CAR expression of different constructs. F, Summary graph from two experiments with three donors included in each experiment (n = 6), illustrating CAR expression of different constructs in primary NK cells. G, Representative diagram from 1 of 3 donors illustrating NK-cell KIR and NKG2A repertoire 24 hours after CAR transfection.
- Figure 2.
Functional analysis of CAR-redirected primary NK cells. A, Representative gating strategy and contour plots to illustrate functional analysis of bulk primary NK cells. B, Summary graphs of six to nine separate experiments (n = 6–12) showing degranulation response against CD19− (P815 and K562) and CD19+ (221.wt and NALM-6) targets in NK cells transfected with HA21 CAR construct. Statistically significant difference between mock- and CAR-transfected cells indicated by black asterisk with underline (n = 12–14). Summary graphs of two separate experiments with three donors (n = 6) showing NK-cell (C) degranulation response and (D) IFNγ+ NK cells against CD19− (P815 and K562) and CD19+ (221.wt and NALM-6) targets across different CAR constructs (n = 15). Summary graphs of nine separate experiments showing (E) degranulation response and (F) IFNγ+ NK cells against CD19− (K562) and CD19+ (221.wt and NALM-6) targets in NK cells transfected with either HA21 or 41BB/ζ CAR construct (n = 12). Statistically significant difference between mock- and CAR-transfected cells indicated by black asterisk with underline. G, Graph showing CAR expression following repeated target or control cells interactions. An arrow indicates when the new pool of target cells was added (n = 4). Experiments were performed three times.
- Figure 3.
CAR-redirected NK cells' response to various cell lines according to their differentiation status. A, Representative gating strategy and contour plots to identify NK cells at various differentiation states and their functional response (CD107a and IFNγ). Summary graphs showing (B) NK-cell degranulation response (data from 14 experiments with two to three donors in each experiment, n = 33) and (C) IFNγ release against CD19− HLA-I–deficient (K562) CD19+ HLA-I low (221) and CD19+ HLA-I+ (NALM-6) according to selected stages of NK-cell differentiation (data from six experiments with two to three donors in each experiment, n = 14). Statistically significant difference between mock- and CAR-transfected cells (all subsets combined) as indicated in the legend.
- Figure 4.
Redirected NK cells harness the functional capacity established by education in its response to CD19+ targets. A, Representative gating strategy to identify single KIR+ educated NK cells and their functional response (CD107a). B, Summary graphs showing degranulation response of mock- or CAR-transfected 2DL3+ (red) and 2DL1+ (blue) NK cells from HLA-C1 or C2 homozygous donors, respectively, against K562, 221, and NALM-6 cells. Statistically significant difference between mock- and CAR-transfected cells indicated by blue and red asterisks for 2DL1 and 2DL3 NK cells, respectively. Statistically significant difference between single 2DL1- and 2DL3-expressing NK cells (without underlines) indicated by black asterisk. Data are aggregated from six experiments (n = 15).
- Figure 5.
CAR-redirected NK cells overcome NKG2A inhibition and boost NKG2C activation of adaptive NK cells. A, Representative contour plots illustrating gating strategy to identify NKG2A or NKG2C single receptor expressing NK cells and their response against 221wt (red) or 221.AEH (blue) targets. Representative contour plots of degranulation of NK cells from selected subsets against the two targets. Numbers represent percentage of the population CD107a+. B, Summary graphs showing CAR-redirected NKG2A (left) NKG2C (right) single positive NK cells' degranulation response against 221.wt or 221.AEH cells. Statistically significant difference between mock- and CAR-transfected cells indicated by black asterisk without underline. Statistically significant difference between 221.wt and 221.AEH cells (with underlines) indicated by black asterisk. Data aggregated from four experiments with two to three donors in each experiment (n = 9). C, Representative graph from one donor illustrating degranulation response from conventional or adaptive NK cells with mock or CAR transfection against 221.wt and 221.AEH cells as well as K562 (CD19– NK-cell target) and P815 (mouse cell line).
- Figure 6.
CAR-redirected NK cells remain sensitive to KIR-mediated inhibition. From HLA-C1C2 heterozygous donors, (A) representative contour plots illustrating gating strategy to identify 2DL1- or 2DL3-expressing NK cells and their response against 221.C1 (red) or 221.C2 (blue) targets. Numbers represent percentage of the population CD107a+. B, Summary graphs showing mock- or CAR-transfected KIR 2DL3 (C1, left) 2DL1 (C2, right) single positive NK cells' degranulation response against 221.C1 or 221.C2 targets. Data aggregated from three experiments with two to three donors in each experiment (n = 7).
- Figure 7.
NK cells exhibit KIR-mediated inhibition to cell line expressing normal amounts of HLA-I. A, Histogram illustrating HLA-ABC expression from Bjab (center) and ROS-50 (right) against normal B cells (left) derived from PBMC. B, Graphs illustrating mock- or CAR-transfected, single KIR expressing CAR-redirected 2DL3 (C1, left) or 2DL1 (C2, right) NK cells' response to Bjab and ROS-50 cell lines. Donors were all HLA-C1C2 heterozygous and the target are both HLA-C2 homozygous. Data are aggregated from three experiments with two to three donors in each experiment (n = 7).
Additional Files
Supplementary Data
- Supplementary Video 1 - Supplementary Video 1. Time-lapse sequence of a serial killer NK cell attacking a cluster of six 221 cells in a 50Ã-50Ã-300 μm3 microwell. The movie plays the same sequence simultaneously side-by-side in fluorescence (left) and bright field (right). The NK cell is shown in blue (in both panels), live targets are shown in green and dead targets in red. At the end of the movie five target cells have been killed. Total imaging time was 4.5 hours with 10 minutes between frames.
Volume 6, Issue 4
