Replicative potential of human natural killer cells

Abstract

The replicative potential of human CD56(+) CD3(-) natural killer (NK) cells is unknown. We found that by exposing NK cells to the leukaemic cell line K562 genetically modified to express 4-1BB ligand and interleukin 15 (K562-mb15-41BBL), they expanded for up to 30 population doublings, achieving numbers that ranged from 1.6 x 10(5) to 1.2 x 10(11)% (median, 5.9 x 10(6)%; n = 7) of those originally seeded. However, NK cells eventually became unresponsive to stimulation and died. Their demise could be suppressed by enforcing the expression of the human telomerase reverse transcriptase gene (TERT). TERT-overexpressing NK cells continued to proliferate in response to K562-mb15-41BBL stimulation for more than 1 year of culture, while maintaining a normal karyotype and genotype. Long-lived NK cells had high cytotoxicity against myeloid and T-lineage leukaemic cells. They remained susceptible to genetic manipulation, becoming highly cytotoxic to B-lineage leukaemic cells after expression of anti-CD19 signaling receptors. Thus, human NK cells have a replicative potential similar to that of T lymphocytes and their lifespan can be significantly prolonged by an increase in TERT activity. We suggest that the methods described here should have many applications in studies of NK cell biology and NK cell-based therapies.

Figures

Figure 1. Replicative potential of human NK cells and their immortalization

(A) NK cells for 3 healthy donors were stimulated with K562-mb15-41BBL cells every 2–3 weeks. Eventually, proliferation ceased and cells died. (B) Cells from 4 healthy donors were stimulated for 1 week with K562-mb15-41BBL cells and then transduced with a retrovirus containing GFP only. GFP-positive cells were stimulated the addition of irradiated K562-mb15-41BBL cells every 2–3 weeks. Proliferation eventually ceased and cells died. (C) Cells from 2 donors transduced as in B but with a vector containing the TERT gene continued to expand more than 150 weeks. (D) TERT mRNA expression by RT-PCR and (E) telomerase activity by TRAP assay in TERT-transduced NK cells.

Figure 2. Immunophenotypic features of TERT-immortalized NK cells

Immortalized NK cells by TERT transduction were analyzed by flow cytometry after 189 days of culture. Broken line histograms represent isotype-matched antibody staining; solid line histograms represent staining with specific PE-conjugated antibodies.

Figure 3. KIR profile of TERT-immortalized NK cells

Immortalized NK cells from 2 donors were analyzed by flow cytometry after 179 days (top row) and 141 days of culture (bottom row). Biexponential density plots show expression of CD16 and of various KIR molecules. Percentage of cells in each quadrant is indicated.

Figure 4. TERT-immortalized NK cells retain their cytotoxicity against NK-sensitive leukemia cell line cells and susceptibility to genetic manipulation

(A) TERT-immortalized NK cells expanded for 24–37 weeks were incubated for 4 hours with myeloid (K562, KG1 and U937) and T lymphoid (Jurkat) leukemia cell lines at the indicated E:T ratios. Each data point represents the mean percentage of duplicate measurements of leukemia cell killing after culture as compared to that of parallel cultures without NK cells. Each assay was done at least twice; representative data is shown. (B) TERT-immortalized NK cells expanded for 45 weeks were transduced anti-CD19-BB-ζ or control vector and incubated with the B-lineage ALL cell lines 380 and RS4;11 as in A. Expression of anti-CD19-BB-ζ expressing cells markedly enhanced cytotoxicity, as previously observed for freshly stimulated primary NK cells.(Imai et al, 2005)

Figure 5. Karyotypic and genetic features of TERT-transduced NK cells

(A) SKY was done for TERT-transduced NK cells from 2 donors. Each panel shows the spectral (RGB) color image (left) and the classified (pseudocolor) chromosome after per-pixel classification of the spectral data (right). Cells maintained a normal karyotype on days 275 and 200, whereas cells collected from late passages had gross genetic changes. Thus, der(6)t(1;6)(q21;q21) and del(16)(q11.1) were found in 15/15 metaphase cells of donor 1 (5 of them had random changes); del(6)(p21.1) and der(16)t(1;16)(q12;q11.2) were found in 15/15 metaphase cells of donor 2. (B) SNP array analysis performed on genomic DNA of early (day 71 and 186) passage TERT-transduced NK cells (day 186 for donor 1 and day 71 for donor 2) did not detect any genomic alteration. Late-passage cells (day 1006 for donor 1 and day 964 for donor 2), had substantial changes (listed in Table 1).