Fig 1.
Comparison of the efficiency of clone formation in the presence of IL-2 and K562-mbIL21 of NK cells at different differentiation and activation stages.
(A) Scheme of NK cell sorting into subsets according to their differentiation and activation stages using CD56, CD57 and HLA-DR markers. (B) Surface HLA-DR expression by CD56bright (upper histogram) and CD56dim (lower histogram) NK cells. Fluorescence minus one (FMO) controls of HLA-DR staining are presented. (C) Analysis of cell subset purity after sorting in the single cell mode. (D) The initial proportions of the discriminated subsets (CD56brightHLA-DR–, CD56brightHLA-DR+, CD56dimCD57–HLA-DR–, CD56dimCD57–HLA-DR+, and CD56dimCD57bright) in total NK cells before sorting of donors whose NK cells were used for clone generation. (E) Clone generation frequencies in the subpopulations, mentioned above, determined in clone collections obtained from six donors. (F) Clone generation frequencies in CD56brightCD57–, CD56dimCD57–, CD56dimCD57bright subsets, recalculated according to the original proportions of HLA-DR+ and HLA-DR−cells in the subsets. Hereinafter significant differences are shown by asterisks as *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001.
Fig 2.
Growing and survival of the NK cell clones.
(A) Dynamics of frequency of living clones derived from NK cell subpopulations of various differentiation and activation states. Data of clone collections from four donors are presented. (B) Percentage of survived clones from the subsets, measured at week 5–8 after the beginning of cultivation. Values are Mean ± SE of four clone collections. (C) Dynamics of cell number in clones derived from different subsets. Data averaged on four clone collections are presented as Mean ± SD. (D) Total cell number registered in long-lived clones (8 weeks and 13 weeks) derived from different subsets.
Fig 3.
Expression of CD56 in NK cell clones.
(A) The expression level of CD56 in NK cell clones obtained from different subsets analyzed 5 weeks after the beginning of cultivation (MFI mean ± SE). (B) CD56 surface expression in a NK cell clone from CD56dimHLA-DR−subset measured at weeks 3 and 5 of cultivation. The dotted line shows autofluorescence, dashed line is the isotype control.
Fig 4.
Expression of CD57 in NK cell clones.
(A) The expression level of CD57 in NK cell clones derived from different subsets, analyzed 5 weeks after the beginning of cultivation (MFI mean ± SE). (B) Proportion of CD57+ cells in NK cell clones from the same donor, obtained from CD57-negative and CD57-positive subsets. (С) Dynamics of CD57 surface expression in short-lived and long-lived clones. Two representative clones for each group are shown. Data on total cell numbers in short-lived (from 5 to 7 weeks) and long-lived (8 weeks and over) clones are presented.
Fig 5.
Surface expression of HLA-DR in NK cell clones.
(A) HLA-DR expression levels in clones derived from different NK cell subpopulations analyzed at week 5 of cultivation. Proportions of HLA-DR+ cells and MFI of HLA-DR+ cells (mean ± SE) of n independent experiments are presented: n = 12 for CD56brightHLA-DR–, n = 9 for CD56brightHLA-DR+, n = 10 for CD56dimCD57–HLA-DR–, n = 13 for CD56dimCD57–HLA-DR+ and n = 11 for CD56dimCD57bright. (B) HLA-DR surface expression in a NK cell clone from CD56dimHLA-DR+ subset measured at weeks 3 and 5. The dotted line indicates autofluorescence, dashed line is the isotype control.
Fig 6.
Surface expression of CD16 in NK cell clones.
(A) Expression levels of CD16 in clones derived from different NK cell subsets analyzed at week 5 of cultivation. Proportions of CD16+ cells and MFI of CD16+ cells (mean ± SE) of n experiments are presented: n = 12 for CD56brightHLA-DR–, n = 10 for CD56brightHLA-DR+, n = 13 for CD56dimCD57– HLA-DR–, n = 13 for CD56dimCD57–HLA-DR+, and n = 8 for CD56dimCD57brigh. (B) CD16 surface expression in a NK cell clone from CD56dimHLA-DR−subset measured at weeks 3 and 5. The dotted line indicates autofluorescence, dashed line is the isotype control.
Fig 7.
Expression of KIR2DL2/DL3 in NK cell clones.
(A) Analysis of KIR2DL2/DL3 expression in clones obtained from different subsets, analyzed after 5 weeks of cultivation. In each subset, the proportion of KIR-positive clones (mean ± SE of 5 independent collections) is presented. In total, KIR2DL2/DL3 expression was analyzed in 139 clones (32 for CD56brightHLA-DR–, 33 for CD56brightHLA-DR+, 24 for CD56dimCD57–HLA-DR–, 25 for CD56dimCD57–HLA-DR+, and 25 for CD56dimCD57brigh cells). (B) Proportions of the subsets in NK cells ex vivo. Mean ± SD of 9 independent experiments is presented. (C) Dynamics of KIR2DL2/DL3 surface expression in an individual NK cell clone. The dotted line indicates autofluorescence, dashed line is the isotype control.
Fig 8.
Surface expression of NKG2A in NK cell clones.
(A) Proportions of CD56bright, CD56dimCD57–, and CD56dimCD57bright subsets in NK cells ex vivo. Mean ± SD of 8 independent experiments is presented. (B) Dynamics of NKG2A surface expression in a NK cell clone from CD56brightHLA-DR+ subset. The dotted line indicates autofluorescence, dashed line–isotype. (C) Analysis of NKG2A expression in clones obtained from different subsets, analyzed after 5 weeks of cultivation. In each subset, the proportion of NKG2A-positive clones is presented (mean ± SE of n clones obtained in 4 clone collections: n = 30 for CD56bright, n = 31 for CD56dimCD57–, n = 19 for CD56dimCD57bright subsets). (D) Analysis of NKG2A expression in clones obtained from CD56dimCD57–/brightNKG2A– subsets, carried out after 5 weeks of cultivation. The proportions of NKG2A-positive clones are presented (mean ± SE of n independent experiments: n = 21 for CD56dimCD57–NKG2A–, n = 19 for CD56dimCD57brighNKG2A– subsets).
Fig 9.
Functional activity of NK cell clones.
(A) IFN-γ production was analyzed by ELISA in 25 clones from one collection: 4 for CD56brightHLA-DR–, 4 for CD56brightHLA-DR+, 4 for CD56dimCD57–HLA-DR–, 9 for CD56dimCD57–HLA-DR+, and 4 for CD56dimCD57brigh cells. (B) Natural cytotoxic activity of clones derived from different NK cell subsets.