Direct contact with the stromal microenvironment induces the development of NK cells from bone marrow progenitors [30
]. Although the importance of IL-15 in this process is undisputed [19
], it is unclear whether IL-15 1) acts on early NK cell precursors, 2) is only needed at the terminal steps of NK cell development or 3) is important role in maintaining lymphocyte proliferation and homeostasis. To investigate this process, we cultured human umbilical cord blood (UCB) CD34+
primitive progenitors on AFT024. We previously demonstrated that AFT024 can support NK cell development and the acquisition of killer immunoglobulin-like receptors (KIR) on CD56+
NK cells when exogenous human cytokines [IL-15, IL-7, IL-3, c-kit ligand (KL), and Flt3-ligand (FL)] are present. We hypothesized that the absence of IL-15 would lead to the accumulation of incompletely-differentiated, progenitor-derived NK precursors at various stages of differentiation. CD56 acquisition was used as a marker of NK cell commitment. While IL-3 or FL alone did not support the development of NK cells, the combination induced 117±32 CD56+
NK cells from 10 starting primitive cells after 4 weeks of culture. These NK cells did not express KIR but CD94 and NKG2A expression could be detected (data not shown). KL significantly increased NK cell development in the absence of exogenous IL-15 but IL-21 had no significant effect. Although AFT024, IL-3 and FL provide the minimally required signals for NK cell differentiation, as expected the number of NK cell progeny was 1–2 log lower in the absence of IL-15 than when IL-15 was included in culture ().
NK cell development on AFT024, IL-3 and FL can occur in the absence of IL-15
In the absence of IL-15, a number of CD56 negative NK cell precursors were defined by the expression of CD34 and CD7 (). These same CD56 negative NK cell precursors were not present in IL-15 containing cultures, presumably because they are short lived under potent NK cell differentiation stimuli of IL-15. The differentiation of CD56 negative precursors (CD34+/CD7−, CD34+/CD7+ and CD34−/CD7+) from primitive CD34+Lin−CD38− progenitors was studied after culture with combinations of IL-3, FL, IL-21, and KL (). After 28 days in culture on AFT024, IL-3 and FL consistently induced outgrowth of all three phenotypic NK cell precursors. Although IL-21 had no independent effect on NK cell commitment in the absence of IL-15 (), the addition of IL-21 to AFT024, IL-3 and FL only increased the CD34+/CD7+ cells (76±13 vs. 130±20, n=15; p <.01; ). In contrast, addition of KL resulted in a net decrease of all three CD56 negative NK cell precursors.
CD56 negative NK cell precursors accumulate in the absence of IL-15
CD56 negative CD34+/CD7−, CD34+/CD7+ and CD34−/CD7+ NK cell precursors were sorted to purity and plated in secondary cultures with fresh AFT024 feeders under NK cell differentiation conditions (IL-15, IL-7, IL-3, KL, and FL). A simultaneously collected control population with the CD34−/CD7− phenotype did not generate NK cells. In contrast, all other precursors gave rise to CD56+ NK cell progeny after secondary culture bearing NK cell receptors (CD94 or KIR) verifying their NK cell lineage (). IL-15 alone was able to generate NK cells from CD34−/CD7+ cells while the more primitive CD34+ NK cell precursors required other cytokines (data not shown). This supports the premise that the CD34−/CD7+ precursor requires IL-15 for final NK cell commitment (defined by the acquisition of CD56).
CD56 negative NK precursors give rise to CD56+ NK cells in secondary culture
Having shown that NK cell precursors can differentiate from human hematopoietic stem cells, we wanted to exclude the possibility that these precursors were artifacts of in vitro culture. For these studies, we evaluated fresh UCB units that provide a source of developing hematopoietic cells. The developmental intermediates were highly enriched in UCB compared to adult peripheral blood from normal volunteers (all P values < 0.0018) for the CD34+/CD7− (31±4.8% vs. 1.9±0.7%), CD34+/CD7+ (3.7±0.8 vs. 0±0%) and CD34−/CD7+ (28±3.9% vs. 0.6±0.4%) population (). These fresh UCB NK cell precursors were studied for their capacity to give rise to CD56+ NK cells (). Fresh UCB CD56 negative NK cell precursors generated more NK cell progeny than did cultured precursors of the same phenotype. IL-15 alone was sufficient to induce further maturation of CD56 negative CD34−/CD7+ precursors to express CD56, CD94 and KIR.
CD56 negative NK cell precursors are markedly enriched in UCB
Fresh UCB NK precursors have different requirements for NK cell differentiation (multiple replicates from 3–6 separates experiments).
CD56− NK cell precursors from fresh UCB were further evaluated for receptors found on mature NK cells. CD16, CD161, CD94, CD159 (NKG2A), CD244 (2B4), NKp44, NKp46 and KIR were not expressed on CD34+ positive cells (data not shown). In contrast, CD34−/CD56−/CD7+ cells variable expressed these NK cell receptors except NKp44 and NKp46, which were not expressed. We found that CD16 (FcRγIII) was expressed on 30±5% of CD34−/CD56−/CD7+ cells and was a good marker to divide the CD34−/CD56−/CD7+ population into cells with distinct phenotypic characteristics (). CD34−/CD56−/CD7+/CD16− cells expressed significantly more CD25, CD62L, CD117 and CD127, markers of immaturity. CD34−/CD56−/CD7+/CD16+ cells were more mature based on their higher relative expression of CD45RA, CD161, CD94, CD159 (NKG2A), CD244 and KIR. These results suggest that the CD34−/CD56−/CD7+ population is heterogeneous and CD16+/CD56− cells may represent a unique stage of NK cell development where NK cell receptor acquisition has already been initiated.
CD7+/CD56− NK cell precursors can be further divided by co-expression of FcRγIII (CD16) (n=8).
Our group has previously shown IL-15 (or IL-2) signals are required for KIR acquisition. However, we had not tested the potential role of other factors. On fresh UCB CD56− NK cell precursors we tested the role of FL and IL-3 on NK cell receptor expression during development. IL-3 had a significant additive effect on the CD34+/CD7− but not CD34−/CD7+ cells to increase acquisition of KIR and NKG2A () suggesting that the effects of IL-3 are on early development. This identifies a novel role for IL-3 and FL as early acting factors that can influence NK cell receptor expression.
Having identified a system to recapitulate early stages of NK cell development, we evaluated a novel murine cell line, designated EL08-1D2, for its capacity to support NK cell development and KIR acquisition. The use of KIR expression in the definition of NK cell maturation has distinguished our stromal-based cultures from those described by others where KIR expression is low or absent under stroma-free conditions [31
]. Under NK cell differentiation conditions (IL-15, IL-7, IL-3, KL, and FL), EL08-1D2 supported NK cell development earlier and more effectively than did AFT024 (). The supportive capacity of EL08-1D2, like AFT024, was most efficient when progenitors were in direct contact with the murine feeder compared to separation by a microporous membrane.
EL08-1D2 is superior to AFT024 to support NK cell differentiation
To definitively understand the differential roles played by the two murine feeders, single CD34+Lin−CD38− cells were cultured on EL08-1D2 and compared to cells cultured on AFT024. The absolute number of NK cells derived from a single primitive progenitor was significantly greater on EL08-1D2 compared to AFT024 after 28 days of culture (123,852±14006 vs. 23143±8117, p=<.0001). Of the 66 EL08-1D2 and 46 AFT024 clones analyzed, 86% of EL08-1D2 cultured cells expressed KIR in a polyclonal pattern compared to 45% on AFT024 (p=<.0001 using Chi Square). The absolute number of KIR+ NK cell progeny from a single primitive progenitor on EL08-1D2 feeders was 3689±801 versus 799±491 when on AFT024 (p=.0027).
The increased NK cell differentiation seen with EL08-1D2 can be explained by its increased ability to support progenitor differentiation, to support proliferation or by a combination of both. This question was addressed by evaluating the NK cell precursors that accumulate in the absence of IL-15. All CD56 negative NK cell precursors are significantly increased in EL08-1D2 supported cultures compared to AFT024 () suggesting that EL08-1D2 exerts its effect on all stages of NK cell development.
EL08-1D2 better supports NK cell precursor differentiation
Even though exogenous huIL-15 was not added to cultures, we questioned whether small amounts of muIL-15 could be produced by mouse stroma, could cross-reactive with human cells and whether the signal could be amplified by trans-presentation with IL-15Rα on the stromal cell surface. Although low level IL-15 transcripts were found on both feeders (data not shown), neither EL08-1D2 nor AFT024 made enough muIL-15 to be detected by ELISA in supernatants collected after 2, 7, or 14 days (data not shown). Flow cytometry showed the absence of muIL-15Rα on AFT024 and a low level of expression on EL08-1D2 (data not shown). To understand the functional significance of these results, we performed additional experiments using a bioassay readout. Purified NK cells, exquisitely sensitive to IL-15, were plated on AFT024 or EL08-1D2. After 14 days of culture in the absence of exogenous huIL-15, no NK cell recovery was seen. Therefore, even if assays for muIL-15 and muIL-15Rα missed some low level of detection, the aggregate functional IL-15 signaling is not enough to support mature human NK survival and likely does not play a significant role in the developmental assays used here.