Fluoroscein isothiocyanate (FITC)-conjugated anti-CD56, anti-CD16, anti-CD33, anti-CD3, anti-CD2, anti-CD11a, anti-CD94, anti-CD80, anti-CD44, anti-granzyme A, Allophycocyanin (APC)-conjugated anti-CD56, anti-CD11c, anti-CD38, anti-CD69, anti-CD117, (Pe)-conjugated anti-CD117, NKB1 (KIR3DL1), anti-CD3, anti-CD16, anti-CD56, anti-perforin, PerCP-conjugated anti-CD3, anti-CD69, anti-CD8 and matching isotype mouse mAbs were purchased from Becton and Dickinson (S Jose, CA). Pe-conjugated anti-CD34, P58.1 (NK2DL1), P58.2(NK2DL2), NKG2A were purchased from Immunotec (Marseille, France). Magnetic beads-conjugated anti-CD56 and mini Macs magnet were purchased from Miltenyi Biotec (Auburn, CA). (APC)-conjugated anti-CD95 and (Pe)-conjugated anti-CD95L were purchased from Caltag (Burlingame, CA). Hyaluronic acid was purchased from Sigma (S Louis, Mo)

CD34+ cells were positively selected from normal donor G-CSF-mobilized peripheral blood stem cells, (PBSC) counted, and frozen in liquid nitrogen until use. All donors gave written informed consent to donate stem cells in NIH protocols 99-H-0046 and 95-H-0049. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-hypaque density separation. Cells were cultured in RPMI 1640 supplemented with 10% AB or 10% FCS serum, glutamine (2 mM) gentamicin, hereafter referred to as complete medium. CD34+ cells were cultured in complete medium, in 24 or 12 or 96 U well plates (Costar), for a minimum of 10 to a maximum of 70 days. Cells were stimulated every 5–7 days with SCF (20–50 ng/ml) (Peprotech, Rocky Hill, NJ) with or without IL-2 (200 U/ml) or IL-15 (1–100 ng/ml). To obtain pure NK populations, CD34+ cells stimulated for 15–21 day with IL-2 were stained with a Pe-conjugated anti-CD56 Moab and CD56+ cells were isolated by electronic sorting using an EPICS ALTRA Flow cytometer (Beckman Coulter, Miami, FL). In some experiments, immature CD56+ cells were selected with an anti-CD56 conjugated magnetic bead column (Miltenyi Inc.). Vigorous mechanical pressure eluted the CD56+cells retained in the column. Peripheral blood NK cells were negatively selected by magnetic sorting using a Miltenyi isolation kit. Positively and negatively selected peripheral blood NK cells were further expanded in vitro as follows: 100 μl of 1 × 10⁵ /ml CD56+ were mixed with 100 μl of 75 Gy irradiated LCL cells in complete medium supplemented with IL-2 (10 U/ml) and 15 % conditioning medium were plated in Costar 96/w round bottom plates. Cells were stimulated every 3 days with CM supplemented with 15% CM for the required time.

In some experiments, cells were stained with Pe-Conjugated anti-CD56 or anti-c-Kit (one color). In other experiments, cells were incubated with FITC-anti-CD56 and Pe-anti-c-Kit (two colors) or a combination of Pe, FITC, PerCP, and APC-conjugated antibodies specific for the desired molecules (four colors). In all cases the cells were stained on ice, for 30 minutes, washed twice, fixed in 1% paraformaldehyde (PFA). For intracellular staining experiments (IC), 1 million cells were first stained with a Pe-conjugated anti-CD56 for 15 minutes at room temperature (RT) in the dark than 2 ml of FACS lysing solution was added to the cell mixture.

After 10 minutes incubation at RT, cells were washed and permeabilized with 0.5 ml FACS perm mix for 10 minutes at RT. Cells were than stained with an anti-Pe-conjugated anti-Perforin Moab and a FITC-conjugated anti-granzyme A moAb or Pe and FITC-conjugated mouse control moAb isotypes for 30 minutes at RT, washed and fixed in 200 ul of 1% PFA. Cells were analyzed by a 4 filters BD FACScan Excalibur. For trans-well experiments, after an initial equilibration (1 hour at 37 C) with complete medium, 200 μl of 5 × 10⁴ - 1 × 10⁵ CD34 cell- derived NK cells or IL-2 activated NK cells cultures were incubated in complete medium in the upper trans-well compartment of a 12-transwell plate (Costar, Cambridge, MA) in the presence or absence of 5 × 10³ - 5 × 10⁴ K562, while 1 ml culture of 5 × 10³K562 were cultured in the lower compartment. A 0.4 μm polycarbonate membrane separated upper and lower compartments. After a 2-day incubation, aliquots of 200 μl of K562 cell culture were removed from the lower compartment and labeled with 3H-TdR. Breaking and washing the transmembrane then permitted harvesting the upper compartment. Cells were counted or labeled with 3H-TdR.

For reverse antibody-directed cell cytotoxicity (ADCC) 6 replicates of 20 μl of effector cells were incubated in a 60-well (40 μl depth) Terasaki plate for 30 m at room temperature in the presence or absence of 5 μl (10μg/ml) 3g8 (anti-CD16). At the same time, FcR+P815 (2 × 10⁶) were incubated in 1 ml of complete medium supplemented with 10 μl of Calcein-AM (Molecular Probes, Junction City, OR) for 30' at 37°C, washed four times and diluted to 1 × 10⁵/ml. After diluting the effector cells, 10 μl of target cells were added, plates were centrifuged and incubated at 37°C for 4 hours. In some experiments, effectors with no mAbs were challenged with K562 cells. A few minutes before scanning the plates using a fluorescent detector 5 μl fluoro-quench was added to each well. The percent of lysis was calculated as follows: 1-(mean test-mean blank)/(mean max-mean blank) ×100.

Total RNA was extracted from 1 × 10⁶ positively selected G-CSF-mobilized peripheral blood CD34+ cells, PBMC, resting or IL-2 activated total peripheral blood CD56+ cells and NK cells using Triazol reagent; complementary DNA (cDNA) first strand was produced using Maloney murine leukemia reverse transcriptase (Roche) with an oligo(dt)^12–18 anti-sense primer (Roche). The cDNA of interest was amplified as shown in table I. Amplified fragments were analyzed in 1.5 % agarose gel electrophoresis in the presence of ethidium bromide (Sigma, S Louis, MO).

The proliferation of K562 and P815 was measured using the tritiated thymidine incorporation (3H-TdR) assay. The first three U-wells of each horizontal row of 96 well plates were filled with 200 μl of negatively selected NK cells or positively selected peripheral blood CD56+ cells, then 100 μl of cultured cells were serially diluted in the remaining wells previously filled with 100 μl of CM. Later, 1 × 10⁴ K562 or P815 cells were added to the cell cultures. After 2 days incubation, cells were pulsed with 1 μCi of 3H-TdR per well (Amersham Biosciences, Piscataway, NJ) Eighteen hours later, 3H-TdR was measured using a beta scintillation counter.

After three weeks stimulation with IL-2 and SCF, G-CSF mobilized peripheral blood CD34+ cells generated CD2-, CD56^high (10.6 ± 11.4) (range 3–39 %) and CD56^low (3.4 ± 2.0) (range 0.7–5 %) in a sample size of 7 normal donors (REF). Some CD56^high cells expressed CD94 and NKG2A but lacked expression of KIR with immunoglobulin like domains KIR2DL2, while CD56^low were KIR negative (Figure 1).

We next evaluated the stage of differentiation of molecules associated with cytotoxicity in immature CD56+ cells. Perforin and granzyme B mRNA expression was measured by RT-PCR in CD34+ cells stimulated with IL-2. Perforin was observed on day 0–1 of culture, but disappeared by day 7. Intracellular staining of immature CD56+ cells revealed a granzyme A content comparable to that found in IL-2 activated PBL (Fig. 2). Notably, the reduction of GAPDH band intensity may reflect loss of viability of the cells upon medium term culture.

We then sought to determine whether the stimulation of CD34+ cells with IL-2 and SCF induced NK natural cytotoxic receptor (NCR) expression. Within two week of culture, immature CD56+ cells expressed some NK activation molecules including CD44, CD69, and CD38 (data not shown) but did not express NCR genes until at least 6 weeks. At than time CD56+ cells expressed NKp46 but not NKp30. Traces of NKG2D and NKP80 RNA expression were also detected by PCR (Fig. 3).

IL-2 activated immature CD56+ cells showed negligible cytotoxicity against K562 compared with the potent lysis exhibited by peripheral blood NK cells. Similarly in a reverse ADCC assay, immature CD56+ cells coated with an anti-CD16 (3g8) showed only low cytotoxicity against the FcR+ cell line, P815, while control peripheral blood NK cells were strongly cytotoxic. We then investigated the effect of CD56+ cells on proliferation of cell lines. Resting CD34+ cells did not inhibit K562 proliferation, while flow sorted, three week cultured CD56+ cells cultures strongly inhibited K562 cell proliferation in a dose dependent manner, reaching 90% inhibition at an E:T ratio of 10:1. The proliferation inhibition induced by immature CD56+ cells ranged between 29 and 96.5 % (Table 2). Immature CD56+ cells also comparably inhibited the proliferation of the NK resistant cell line P815, indicating that the proliferation inhibition was independent of the resistance of this line to NK-mediated cytotoxicity. Unlike immature CD56+ cells, peripheral blood-derived CD56+ cells proliferated in the presence of IL-2. Nevertheless the proliferation attributable to K562 cells was also abolished when cultured with CD56+ cells (Fig. 4). Trans-well experiments were used to determine whether the inhibition of K562 cell proliferation was mediated by cell-interaction or by soluble factors. In three experiments, the average 3H-TdR incorporation of K562 cells when separated by a membrane from immature CD56+ cells was 5361 ± 1967 versus 5110 ± 1539 cpm for K562 growth in the absence of CD56+ soluble factors. This suggested that proliferation was primarily blocked by cell-cell contact (Table 3). To further examine the mechanism of K562 proliferation inhibition, we cultured K562 with either magnetically sorted immature CD56+ cells in a transwell upper chamber and K562 cells in the lower chamber for two days. Viable K562 cells (> 95% trypan blue negative cells) were recovered from immature CD56+ cells but not from mature CD56+ cultures. After depleting the K562/immature CD56+ cultures of CD56+ cells using antibody-coated magnetic beads, residual K562 cells again proliferated, indicating that in the absence of cytotoxicity, proliferation inhibition was reversible (data not shown).