Figure 1.
NK cell production from fresh and frozen CBCD34+.
(A) Total fold expansion and (B) CD3−CD56+ NK cell number of fresh (n = 3) and frozen (n = 4) CBCD34+ cultures at different time points. (C) Intracellular expression of IFN-γ and (D) secreted IFN-γ as measured by ELISA for NK cells from fresh (n = 3) and frozen (n = 4) CBCD34+ cultures. (E) Degranulation assay using CD107a on CD56+CD3− cells from fresh (n = 3) and frozen (n = 4) CBCD34+ cultures. NK cells from fresh (n = 3, solid line) and frozen (n = 4, dotted line) CBCD34+ cultures were co-incubated with 51Cr-labeled K562 cells (F) or 51Cr-labeled P815 cells (G) coated with anti-CD16 or an isotype control at different effector-target ratios in a standard 4 h 51Cr-release assay. The statistical analysis was performed using Mann-Whitney test, * P<0.05, ** P<0.005.
Figure 2.
NK cell production from CBCD34+ and PBCD34+.
(A) Total fold expansion and (B) CD3−CD56+ NK cell number of CBCD34+ (n = 8) and PBCD34+ (n = 6) cultures at different time points. (C) Representative plot of CD56 vs CD16 from the lymphocyte gate for CBCD34+ and PBCD34+ cultures. (D) Representative side scatter vs CD56+ plots for CBCD34+ (upper row) and PBCD34+ (bottom row) cultures at days 7, 14, 21, 28, 35 and 42. Mann-Whitney test was performed, * P<0.05, ** P<0.005.
Figure 3.
NK cell development in CBCD34+ and PBCD34+ cultures.
NK cell stages 1–4 for one representative sample from CBCD34+ (n = 8) (A) and PBCD34+ (n = 6) (B) cultures. Percentages are gated from CD3− cells according to the following NK cell stages: stage 1: CD34+CD117−CD94−, stage 2: CD34+CD117+CD94−, stage 3: CD34−CD117+CD94− and stage 4: CD34−CD117+/−CD94+. (C) NK cell stages are shown for one representative sample for CBCD34+ (n = 8, upper panel) and PBCD34+ (n = 6, bottom panel) cultures at different time points. Stage 1 and 2 are from the CD3−CD94− gate and stages 3 and 4 from the CD3−CD34− gate. Transcriptional analysis for each time point is shown for transcription factors involved in NK cell differentiation (left panel) and maturation (right panel) for CBCD34+ (n = 4) (D) and PBCD34+ (n = 3) (E) cultures. Values are normalized using three reference genes. Higher ratio values correspond to less mRNA expression.
Figure 4.
Phenotype of NK cells differentiated from CBCD34+ and PBCD34+ cultures.
(A) Surface antigens were detected by flow cytometry. A representative sample for the expression of each antigen in the CD56bright and CD56dim subsets for CBCD34+ and PBCD34+ cultures is shown. (B) Transcriptional analysis for KIR2DL1 and KIR2DL2 at days 21, 28 and 35 is shown for NK cells from CBCD34+ (n = 4) and PBCD34+ (n = 3) cultures. Values are normalized using three reference genes. (C) Graphs depict the surface expression at day 35 of IL-12β1 and CXCR4 on NK cell subsets from frozen CBCD34+ (n = 4–5) and PBCD34+ (n = 3) cultures. Mann-Whitney test was performed, * P<0.05, ** P<0.005.
Figure 5.
Killing capacity and cytokine production by NK cells from CBCD34+ and PBCD34+ cultures.
(A) TNF-α and IFN-γ secretion by NK cells was measured by ELISA and intracellular expression of IFN-γ was detected by flow cytometry for CBCD34+ (n = 4) and PBCD34+ (n = 5) cultures. (B) CD107a degranulation assay using non-stimulated CD56+CD3− cells or cells stimulated with K562, Raji or PMA&Iono for CBCD34+ (n = 9) and PBCD34+ (n = 6) cultures. (C) Representative intracellular staining for perforin and granzyme B at day 35 in NK cells from CBCD34+ (n = 9) and PBCD34+ (n = 6) cultures. The right panel shows granzyme B mRNA levels at days 21, 28 and 35 in NK cells from CBCD34+ (n = 5) and PBCD34+ (n = 3) cultures. (D) NK cells from CBCD34+ (n = 6) and PBCD34+ (n = 4) cultures were co-incubated with 51Cr-labeled K562 cells or 51Cr-labeled P815 cells coated with anti-CD16 or an isotype control at different effector-target ratios in a standard 4 h 51Cr-release assay. The statistical analysis was performed using Mann-Whitney test. * P<0.05, ** p<0.005.
Figure 6.
Killing of K562 in vivo by NK cells from CBCD34+ and PBCD34+ cultures.
NSG mice were injected with GFP-K562 cells followed by CBCD34+-NK cells or PBCD34+-NK cells 24 h later. (A) Percentage of GFP-K562 cells detected in the BM, liver, lungs and spleen of NSG mice. (B) Percentage of NK cells detected in the BM, liver, lungs and spleen of NSG mice. The statistical analysis was performed using Mann-Whitney test. * P<0.05.
Figure 7.
Expression of the myeloid marker CD33 and the effect of blocking CD33 on the differentiation of NK cells from CBCD34+ and PBCD34+.
(A) Percentage of CD45+CD33+ cells at different time points for CBCD34+ (n = 8) and PBCD34+ (n = 6) cultures. (B) FACS plots showing CD33 expression against CD56 at different time points for CBCD34+ (n = 8) and PBCD34+ (n = 6) cultures. NK cells from CBCD34+ (n = 4–6) or PBCD34+ (n = 3–5) were incubated in the presence or not of a CD33 blocking antibody. Secreted IFN-γ (C), CD107a degranulation (D), intracellular IFN-γ (E) and NK cell killing in vitro of the malignant cell line K562 in a 10∶1 effector-target ratio (F) were evaluated. Statistical analysis was performed using paired T-test. * P<0.05.
Figure 8.
Effects of IL-12 stimulation on the function of the differentiated NK cells.
NK cells were incubated with IL-12 for 4 h, 24 h or 40 h. (A) The figure illustrates the effect of IL-12 on the secretion of IFN-γ and (B) TNF-α measured by ELISA after incubation with PMA&Iono. (C) The graph depicts the intracellular expression of IFN-γ after incubation with PMA&Iono. (D) The graph shows CD107a degranulation after incubation with PMA&Iono. (E) NK cell killing capacity against 51Cr labeled K562 cells or (F) P815 cells coated with anti-CD16. The effector-target ratio used was 10∶1. Statistical analysis was performed using Mann-Whitney test. * P<0.05, ** p<0.005.