Activated Notch Supports Development of Cytokine Producing NK Cells Which Are Hyporesponsive and Fail to Acquire NK Cell Effector Functions

Notch1 induces CD7 on CD34⁺ UCB progenitors. UCB CD34⁺ progenitors were transduced in Fibronectin-coated transwells in the presence of c-kit-L, Flt3-L, IL-7, and TPO with MSCV-based eGFP and ICN vectors, FACS sorted for CD34⁺eGFP⁺ cells, plated in contact with murine embryonic liver stroma cell line EL08-1D2 with IL-3, c-kit-L, Flt3-L, IL-7, and IL-15, and cultured for 28 days. (A) The induction of specific mRNA from cultured ICN⁺ cells (■) and eGFP⁺ cells was analyzed by Q-RT- PCR. Relative transcripts of Notch1 (ICN) and downstream ICN targets Hes1 and Hes5 are relative to the level of GAPDH as a control for RNA integrity and reported as a ratio to eGFP controls. Bars represent mean expression ± SEM of 3 independent experiments. (B) ICN⁺ and eGFP⁺ control cells were analyzed 2 days after transduction or after culture with EL08-1D2 stroma and cytokines (IL-3, c-kit-L, Flt3-L, IL-7, and IL-15) for 7 additional days. All flow cytometry plots were gated on eGFP⁺ lymphocyte gate. Only ICN⁺ cell gave rise to a population of CD34⁺CD7⁺cells, which subsequently differentiated into CD34⁻CD7⁺ (and predominantly CD56⁻) lymphoid precursors.

Activated Notch1 induced early expression of CD45RA and the adhesion marker L-selectin (CD62L). Expression of CD62L and CD45RA on fresh CD34⁺ UCB progenitors is shown in the top row. CD34⁺eGFP⁺ and CD34⁺ICN⁺ progenitors cultured with EL08-1D2 stroma and cytokines (Flt3-L, IL-7, c-kit-L, IL-15, and IL-3) for 7 days exhibited higher L-selectin and CD45RA expression compared to CD34⁺eGFP⁺ (representative of 4 independent experiments).

Notch1 enhances early NK cell commitment from CD34⁺ UCB precursors. EL08-1D2 stroma and cytokine (Flt3-L, IL-7, c-kit-L, IL-15, and IL-3) cultured cells were harvested, counted, and analyzed by flow cytometry. (A) Absolute numbers of cultured eGFP⁺(▴) and ICN⁺(▴)-derived CD56⁺CD3⁻ NK cells calculated per 100 plated cells were compared at weekly intervals. In the presence of stroma, activated Notch1 conferred an early growth advantage to cultured cells compared to eGFP⁺ control cells (▴) at days 14 and 21 (∗P = .005; mean of 3 independent experiments shown). (B) Stroma cultured eGFP⁺ cells and ICN⁺ cells were harvested at day 28 and compared for expression of CD56 and CD7 (gated on CD56⁺CD3⁻ cells). CD7 was highly expressed only on ICN⁺ cells (representative of 6 experiments shown).

CD34⁺ICN⁺-derived NK cells are hyporesponsive and exhibit immature function. (A) ICN⁺ and eGFP⁺-derived cells were cultured with EL08-1D2 stroma and cytokines (Flt3-L, IL-7, c-kit-L, IL-15, and IL-3). Stroma cultured CD34⁺-derived cells were harvested at day 28 and compared in ⁵¹Cr release cytotoxicity assay against K562 targets at various E:T ratio as shown. ICN⁺-derived cells (▴) showed a significant reduction in cytotoxicity compared to eGFP⁺ cells (●) (mean of 7 independent experiment ± SEM shown; ∗P < .05). (B) Cultured CD34⁺ICN⁺ and CD34⁺eGFP⁺ cells were harvested at day 28 and suspended in concentration 1 × 10⁶ cells/mL. Interferon-γ production was analyzed by intracellular stain following stimulation with IL-12 (10 ng/mL) and IL-18 (100 ng/mL) for 18 hours, followed by Brefeldin A in the last 5 hours of the incubation and permeabilization using cytofix/cytoperm (4 independent experiments ± SEM; ∗P < .05). (C) Chemokines and cytokines were measured by Luminex from eGFP⁺ and ICN⁺ cell-free supernatants prepared after stimulation of 10⁶ cells/well with PMA/ionomycin. Significant differences between eGFP⁺ (□) and ICN⁺ (■) cell-free supernatants were seen for IL-13, GM-CSF, and CCL2 (MCP-1) and IL-2 (mean of 5 independent experiments with SEM is shown; ∗P < .05).

Expression of NK cell receptors is altered by activated Notch. (A) Representative histograms of NK cell receptors on NK cells derived from CD34⁺eGFP⁺ and CD34⁺ICN⁺ cells coculured with EL08-1D2 stroma and cytokines (Flt3-L, IL-7, c-kit-L, IL-15, and IL-3) are shown (gated on CD56⁺CD3^- cells). ICN⁺ NK cells demonstrated less NKG2A and KIR expression. (B) Bars represent the average expression of receptors on CD56⁺CD3⁻ NK cells derived from stroma cultured CD34⁺eGFP⁺ (□) and CD34⁺ICN⁺ (■) at day 28 (mean of 4 independent experiments ± SEM). (C) CD34⁺eGFP⁺ and CD34⁺ICN⁺ cells were cultured for 28 days on EL08-1D2 with Flt3-L, IL-7, c-kit-L, and IL-3 with and without IL-15. In the absence of IL-15, only CD34⁺ICN⁺ population gave rise to NK cells. NKG2A expression on CD34⁺ICN⁺-derived NK cells is compared to eGFP-derived NK cells with and without IL-15.

Notch 1 can replace stroma in supporting NK cell commitment from CD34⁺ UCB progenitors. (A) NK cells derived from CD34⁺eGFP⁺ and CD34⁺ICN⁺ progenitors were cultured without stroma but with cytokines (Flt3-L, IL-7, c-kit-L, IL-15, and IL-3). Absolute numbers of cultured eGFP⁺ (♦) and ICN⁺ (▴)-derived CD56⁺CD3⁻ NK cells are shown. Only CD34⁺ICN⁺ cells were able to expand without stroma (mean of 3 independent experiments shown). (B) The phenotype of CD34⁺ICN⁺-derived stroma-free cultured NK cells at day 28 is shown, which indicates the accumulation of CD56⁺CD117^high CD94^lo NKG2A^locells.

Activated Notch induced a generation of cyCD3⁺ lymphocytes capable of producing IL-2. UCB CD34⁺ progenitors transduced with GFP and ICN were cultured and analyzed at day 28. (A) ICN⁺ or eGFP⁺-derived cells were incubated in the presence of phorbol ester and calcium ionophore (PMA/ionomycin) for 5 hours. Brefeldin A was added to cultures and cells were permeabilized using cytofix/cytoperm. Representative flow cytometry plots for expression of CD56 and cytoplasmic CD3 (cyCD3) are shown (left panel). The cyCD3⁺ICN⁺ population was capable of IL-2 production after PMA stimulation (right panel, representative of 4 independent experiments). (B) RNA transcripts of RAG1, RAG2, pre-Tα, GATA3, cyCD3 γ, δ, ε, zeta were analyzed by QRT-PCR and compared between ICN⁺ and eGFP-derived cells harvested at day 28. The induction of specific mRNA is relative to the level of GAPDH expression and is normalized to the ratio of eGFP-derived NK cells. Bars represent the average of 3 independent experiments ± SEM.