Potential mechanisms of human natural killer cell expansion in vivo during low-dose IL-2 therapy

The continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4–6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G₂/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34⁺ bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56⁺CD3⁻ NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2–dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.

Natural killer (NK) cells are large granular lymphocytes that secrete IFN-γ and demonstrate spontaneous cytotoxic activity against malignant and virally infected cells (1, 2). All human NK cells express the CD56 antigen, an isoform of the neural cell adhesion molecule. Approximately 10% of NK cells have high-density expression of CD56 (CD56^bright) on their surface and constitutively express both the high-affinity heterotrimeric IL-2 receptor (IL-2Rαβγ) and the intermediate-affinity heterodimeric IL-2Rβγ (3, 4). Ninety percent of human NK cells have low-density expression of CD56 (CD56^dim) and only express the intermediate-affinity heterodimeric IL-2Rβγ (2). In vitro, picomolar (pM) concentrations of IL-2 bind to the high-affinity IL-2R expressed on CD56^bright NK cells, resulting in a strong proliferative response that is not seen in CD56^dim NK cells or in resting T cells in the absence of other mitogenic signals (3).

The in vivo administration of low-dose IL-2 by prolonged continuous intravenous infusion or daily subcutaneous injection results in pM concentrations of IL-2 that selectively expand CD56^bright NK cells with little change in CD56^dim NK or T-cell numbers (5–10). The mechanism by which this expansion occurs in vivo is unknown. We hypothesized that activation of CD56^bright NK cells via the high-affinity IL-2Rαβγ led to a proliferative expansion of the population in vivo, similar to that seen in short-term culture in vitro (3, 4). However, our preliminary in vitro analysis did not support this hypothesis (5). In the current report, we analyze human CD56^bright NK-cell proliferation, apoptosis, and differentiation in vitro and perform cell-cycle analysis on human CD56^bright NK cells undergoing expansion during low-dose IL-2 administration in vivo. We demonstrate that selective expansion of NK cells during prolonged exposure to low concentrations of exogenous IL-2 does not appear to result from mature NK-cell proliferation in vivo. Rather, the in vivo expansion likely results from an IL-2–dependent decrease in NK-cell death and an increase in NK-cell differentiation from hematopoietic progenitor cells (HPCs).

Proliferative response of CD56^bright NK cells to long-term culture with rIL-2. The continuous in vivo infusion of low-dose IL-2 results in pM serum concentrations of IL-2 and significant, selective expansion of CD56^bright NK cells, but only after 4–6 weeks of therapy (Table 1) (5). CD56^bright NK cells exhibit a brisk proliferative response to pM concentrations of rIL-2 during short-term (72–96 hours) culture in vitro (3, 4), but proliferation in extended cultures have not been reported. CD56^bright NK cells were therefore cultured for 12 days in medium containing 100 pM rIL-2, which was replaced every 72 hours. In repeated experiments, the CD56^bright NK-cell population initially exhibited a substantial increase in ³H-TdR incorporation that peaked at 4–6 days, followed by a gradual decline to baseline levels on day 10 (Figure 1a). Consistent with the ³H-TdR incorporation, cell staining with Hoechst dye showed less than 2% of CD56^bright NK cells in cycle on day 1 (data not shown), but more than 20% of CD56^bright NK cells in the G2/M phase of the cell cycle after day 4 of culture in rIL-2. Notably, 42–59% of CD56^bright NK cells were found within the hypodiploid peak, characteristic of cells undergoing programmed cell death (Figure 1a, inset). Resting T cells or CD56^dim NK cells do not proliferate in response to 100 pM IL-2 alone (3).

Figure 1

(a) 12-day ³H-TdR incorporation of CD56^bright NK cells cultured in 100 pM rIL-2. Freshly isolated CD56^bright NK cells (>97% pure) were plated in 96-well plates and cultured in medium containing 100 pM rIL-2, as described in Methods. Cells were assayed for ³H-TdR incorporation every 48 hours. Each time point represents the mean cpm ± SEM of triplicate wells. Inset: Histogram of Hoechst-stained CD56^bright NK cells cultured for 4 days with medium containing 100 pM rIL-2. Cell number is measured along the ordinate, DNA content along the abscissa. Five thousand events were collected and analyzed. Number above the region marker (22%) indicates the percentage of cells in the G2/M phase of the cell cycle. Approximately 45% of cells were found in the hypodiploid region of the histogram, usually indicative of apoptosis. (b) Enumeration of CD56^bright NK cells by vital dye exclusion during 12-day culture in medium supplemented with or without 100 pM rIL-2. Values shown represent the mean ± SEM of readings done in triplicate wells. This figure is representative of four independent experiments performed on sorted CD56^bright cells from four normal individuals.

Table 1

Cell-cycle analysis of lymphocyte subsets before and during low-dose IL-2 therapy

Cell-enumeration assays were performed by vital dye exclusion on days 2, 6, and 12 of culture to assess further the fraction of IL-2–responsive CD56^bright NK cells. No CD56^bright NK cells cultured in the absence of rIL-2 survived after day 6 (Figure 1b). In contrast, cells cultured in the presence of 100 pM rIL-2 showed a 60 ± 4.3% decrease in number during the first 6 days of culture, but no significant decrease thereafter up to day 12. Thus, the significant decline in ³H-TdR incorporation seen in CD56^bright NK cells between days 6 and 12 of culture in rIL-2 was not accounted for by a continual decrease in absolute cell number or increase in cell death. Indeed, the vast majority of cell death after IL-2 activation occurs during the first 6 days of culture when proliferation is maximal (Figure 1). The hypodiploid nuclei seen with Hoechst staining during the first 6 days of culture (inset, Figure 1a) depicts cleaved DNA, characteristic of apoptosis (14). This was confirmed with direct visualization of DNA by agarose gel electrophoresis, which demonstrated DNA laddering that is characteristic of apoptosis (data not shown). Thus, although a fraction of CD56^bright NK cells were proliferating during the first 6 days of culture, a fraction of CD56^bright NK cells were also undergoing programmed cell death.

We next examined whether the CD56^bright NK cells undergoing proliferation in response to rIL-2 were also the cells undergoing programmed cell death. Cells transiting the cell cycle incorporate the thymidine analog BrdU in a manner proportional to the number of times the cell has passed through the cell cycle. Simultaneously, cellular DNA content can be evaluated by PI staining. Apoptotic nuclei stained with PI appear on the red fluorescence channel as a population showing hypodiploid DNA that is easily distinguished from the normal diploid or hyperdiploid peak of viable cells (16). Figure 2a depicts a control experiment in which CD56^bright cells were analyzed after 6 days of culture in medium containing 10% HAB without IL-2. When performed with CD56^bright NK cells incubated in the presence of 100 pM IL-2 for 6 days, a fraction of cells that remained quiescent during the assay were within the hypodiploid or apoptotic fraction (Figure 2b). None of the cells incorporating BrdU and therefore transiting the cell cycle were within the hypodiploid fraction. Thus, a fraction of CD56^bright cells that were unresponsive to the IL-2 proliferative signal were also undergoing programmed cell death and responsible for the drop in cell number seen during early cell culture in IL-2. This suggested that the CD56^bright NK cells originally proliferating in response to IL-2 during the first 6 days of culture made up the majority of cells that remained alive yet quiescent during the extended (i.e., 12-day) cell culture (Figure 1). The CD56^bright NK cells that survived without cycling during the extended cell culture required IL-2 binding to the high-affinity IL-2Rαβγ to prevent programmed cell death, because the addition of neutralizing anti–IL-2 polysera or anti–IL-2Rα mAb resulted in greater than 90% cell death by day 10 in repeated experiments (data not shown).

Figure 2

Correlation of apoptosis and proliferation of IL-2–stimulated CD56^bright NK cells as evaluated by PI/BrdU assay. Shown are representative contour plots of sorted CD56^bright NK cells cultured in the absence (a) or presence (b) of 100 pM IL-2 for 6 days and assayed as described in Methods. BrdU incorporation (proliferative activity) is measured along the abscissa (FITC fluorescence). PI fluorescent intensity (DNA content) is measured along the ordinate (red fluorescence). Cells that have undergone apoptosis show a hypodiploid complement of DNA and fall below the dashed horizontal divider. Normal (viable) diploid cells are found between the dashed and solid horizontal lines. In the absence of IL-2 (a), there is no evidence of BrdU incorporation, and the vast majority of CD56^bright NK cells are found within the hypodiploid fraction by day 6. In the presence of IL-2 (b), a fraction of cells proliferate, incorporate BrdU, and are therefore seen to the right of the vertical divider. Apoptosis has only occurred within the nondividing fraction of cells (left, below dashed line). This is representative of three independent experiments with similar results.

CD56^bright NK cells expanded in culture are functional innate immune effector cells. We next assessed the function of CD56^bright NK cells that were cultured for 12 days in the presence of IL-2. IL-2 (1 nM) plus IL-12 (10 U/mL) was added to these otherwise unmanipulated NK-cell cultures on days 2, 6, and 12, and IFN-γ production was measured in culture supernatants 2 days after the addition of IL-12 (Figure 3a). NK cells cultured in the presence of 100 pM IL-2 maintained the ability to produce IFN-γ throughout the 12-day period of culture in response to IL-2 + IL-12, whereas those cultured in medium alone had no IFN-γ production in response to IL-2 + IL-12. As another measure of viable NK-cell function, we also evaluated cytotoxic function throughout a 12-day culture in the presence or absence of 100 pM IL-2. Again, CD56^bright cells cultured in the presence of IL-2 maintained significant cytotoxic activity, whereas those cultured in the absence of IL-2 did not display any ability to lyse target cells (Figure 3b).

Figure 3

CD56^bright cells cultured in 100 pM IL-2 maintain functional capacity relative to culture in medium alone. (a) NK-cell production of IFN-γ in response to stimulation with IL-2 plus IL-12. Twenty thousand CD56^bright cells per well were plated in the presence or absence of 100 pM IL-2. On days 2, 6, and 12, IL-2 (1 nM) and IL-12 (10 U/mL) were added, and 2 days later, the supernatants were collected and analyzed for IFN-γ by ELISA. Results depict the mean ± SEM for triplicate wells. (b) Cytotoxicity of CD56^bright cells against K562 targets after 2, 6, or 12 days of culture with or without 100 pM rIL-2. Values given represent the mean ± SEM of readings from triplicate wells for each time point, at an effector-to-target ratio of 5:1. The data shown are representative of three independent experiments.

CD56^bright NK cells expanded in vivo are not transiting the cell cycle. We performed cell-cycle analysis on lymphocyte subsets from five patients receiving a continuous in vivo infusion of low-dose IL-2 therapy. For these five patients, there was a significant (i.e., 7- to 36-fold; P < 0.01) and selective in vivo expansion of CD56^bright NK cells during low-dose IL-2 therapy. Despite this, there was no difference in the percent of CD56⁻ cells (i.e., T cells and B cells), CD56^dim NK cells, or CD56^bright NK cells traversing the G2/M phase of the cell cycle during low-dose IL-2 therapy. Further, patients receiving IL-2 showed no increase in the fraction of cells entering cell cycle compared with four similar patients not receiving IL-2 (Table 1). A graphical depiction of the selective NK-cell expansion and a histogram illustrating the fraction traversing the G2/M phase of the cell cycle from a representative patient receiving low-dose IL-2 therapy are shown in Figure 4. Thus, like the CD56^bright NK cells studied in vitro, the CD56^bright NK cells exposed to a prolonged continuous infusion of exogenous IL-2 in vivo are in a quiescent phase of the cell cycle. Hence, the IL-2–induced increase in the absolute number of CD56^bright NK cells in vivo does not appear to result from continued proliferation of mature CD56^bright cells in peripheral blood.

Figure 4

CD56^bright cells expanding in vivo to IL-2 are not transiting the cell cycle. (a) Dot plot of a representative patient’s lymphocytes stained with anti–CD56-PE and analyzed by flow cytometry during low-dose IL-2 therapy. Percentages located within individual gated regions indicate the fraction each subset comprises as a percentage of total cells analyzed. PE fluorescent intensity is given along the ordinate, FITC fluorescent intensity along the abscissa. Hoechst cell-cycle analyses of CD56^bright NK cells (b), CD56^dim NK cells (c), and CD56^negative, i.e., T and B cells (d) represented in a, are shown in the right column. Cell number is measured along the ordinate, DNA content along the abscissa. Percentages noted above the region markers of each subpopulation indicate the percentage of cells in the G2/M phase of the cell cycle. A summary of data for five patients during IL-2 therapy is found in Table 1.

Low-dose IL-2 promotes the generation of CD56^bright NK cells from CD34⁺ HPCs in vitro. The data presented thus far suggested that an IL-2–dependent delay in NK-cell death, rather than enhanced NK-cell proliferation, was contributing to the expansion of CD56^bright NK cells during low-dose IL-2 therapy (Table 1). However, we found that mature T cells, which do not expand in patients receiving low-dose IL-2 therapy, showed prolonged survival similar to NK cells when cultured with 100 pM IL-2 in vitro (data not shown). We therefore investigated what role, if any, exogenous administration of low-dose IL-2 might have on the differentiation of CD56^bright NK cells from bone marrow–derived HPCs. When purified CD34⁺ HPCs were cultured in medium containing 10% human serum and 100 pM rIL-2 for 21 days, there was a striking appearance of CD56^brightCD3^– NK cells compared with cells cultured in 10% human serum alone (Figure 5, top panels). These cells had large granular lymphocyte morphology and demonstrated potent NK cytotoxic activity (data not shown). Further, when low concentrations of IL-2 were combined with the c-kit ligand, a hematopoietic growth factor known to be abundantly expressed by bone marrow stromal cells and measurable in normal human serum (19), there was fourfold greater expansion of CD56^brightCD3^– NK cells from CD34⁺ HPCs, compared with cells cultured with exogenous IL-2 alone (Figure 5, middle panels). Thus, in addition to maintaining survival of CD56^bright NK cells, low concentrations of exogenous IL-2 can enhance CD56^bright NK-cell differentiation from CD34⁺ HPCs in vitro.

Figure 5

Effect of low-dose IL-2, KL, or IL-2 plus KL on the development of NK cells from CD34⁺ HPCs. Upper panels show phenotypic evaluation of cells produced from 21-day culture of CD34⁺ HPCs with 100 pM IL-2, 7 nM KL, or a combination of both. Anti–CD3-FITC and anti–CD56-PE mAb’s that were subsequently analyzed by FACS are shown. Graphs below show absolute numbers of total mononuclear cells or CD56⁺ cells produced by CD34⁺ HPC cultures. Absolute numbers of CD56⁺ cells were obtained by multiplying absolute numbers of cells counted in trypan blue by percent CD56⁺ cells obtained by FACS. The data are representative of six separate experiments.

We examined the potential mechanisms by which human NK cells selectively expand during prolonged continuous in vivo infusions of low-dose IL-2. Earlier work had shown that the CD56^bright NK-cell subset is unique among resting lymphocytes in its constitutive expression of the high-affinity IL-2R and its brisk proliferative response to IL-2 in short-term culture (3, 4). However, the extent of CD56^bright proliferation beyond 4 days of culture and characterization of CD56^bright expansion in vivo has not been reported for patients receiving this therapy.

The data presented in this report show that the CD56^bright NK-cell response to pM concentrations of IL-2 is heterogeneous. More than half of these freshly isolated cells undergo apoptosis in the absence of proliferation, despite the presence of IL-2. Thus, a sizable subset of these cells must be considered proliferatively unresponsive to this cytokine. The proliferative response induced by low-dose IL-2 that was seen in the remaining CD56^bright NK cells was limited and was followed by an exit from the cell cycle in the absence of additional stimulation. Although there is evidence to suggest that proliferating lymphocytes are susceptible to apoptosis (17, 20), the data presented here show that it was the proliferating cells that were rescued from cell death. The reason for the halt in proliferation is not clear, but was due neither to the absence of IL-2 nor the lack of a functional IL-2Rαβγ. Indeed, if IL-2 was neutralized or if the high-affinity IL-2R was blocked, the majority of CD56^bright NK cells became susceptible to programmed cell death in prolonged culture. Thus, expression of the high-affinity IL-2Rαβγ was required in both processes, i.e., initiating growth and sustaining subsequent survival, and the functional responses to IL-2 changed over time. The gradual loss of proliferation may involve the cells’ inability to replenish intracellular intermediates critical for IL-2–induced proliferation, and/or the cells’ progressive differentiation in the presence of IL-2. In contrast to proliferation, NK-cell survival could be maintained with IL-2 for weeks. IL-2 preserved NK-cell nuclear membrane and functional integrity, as evidenced by measurable cytokine production and cytotoxic activity after extended in vitro culture. However, low concentrations of IL-2 had a similar effect on preventing T-cell death in vitro. Thus, the IL-2–mediated delay in cell death observed in vitro cannot alone account for the selective in vivo expansion of CD56^bright NK cells during prolonged IL-2 therapy.

Lymphocytes obtained from five patients receiving a prolonged continuous infusion of IL-2 were studied for evidence of proliferation. NK cells examined directly without in vitro culture did not show an increased fraction in the G2/M or S phase of the cell cycle, relative to T cells obtained at the same time from the same patients. NK cells obtained from patients before low-dose IL-2 therapy and from comparable patients not receiving IL-2 had similar cell-cycle profiles. This suggests that IL-2–induced proliferation of CD56^bright NK cells is not responsible for the observed expansion in vivo. Although we cannot be certain that some proliferation of NK cells may occur during the first 6 days of in vivo IL-2 infusions, significant in vivo NK-cell expansion does not occur until 4–6 weeks after initiation of IL-2 (5).

The failure to detect enhanced NK-cell proliferation in vivo during IL-2 therapy leaves two other hypotheses to account for the observed alteration in NK-cell homeostasis. First, exogenous administration of IL-2 could delay NK-cell death in vivo. Indeed, patients’ NK cells that were expanded in vivo could be sustained in vitro with 100 pM IL-2 and 10% human serum. This effect appears to be the result of IL-2’s ability to prevent NK-cell apoptosis. However, this effect can also be seen with T cells, arguing against this as the only mechanism responsible for selective CD56^bright NK-cell expansion. Increased NK-cell production via differentiation from BM precursors is a second, alternative mechanism explaining the selective NK-cell expansion in vivo. Indeed, studies have previously shown that relatively high concentrations of IL-2 can support NK-cell differentiation from CD34⁺ BM HPCs in vitro (21–24). In this report we show that BM-derived CD34⁺ HPCs can differentiate into CD56^bright NK cells with 100 pM rIL-2 in vitro, without a stromal cell layer. This NK-cell differentiation was significantly enhanced by KL, a factor produced by BM stroma that is normally measurable in human serum (19). Additional in vitro (25) and in vivo (26) evidence suggests that endogenously produced KL and Flt3 ligand are likely to enhance IL-2–induced NK-cell differentiation from HPCs. Indeed, we have recently shown that KL or flt3 ligand induces a distinct CD34^brightIL-2Rβ⁺ NK-cell progenitor from CD34⁺ HPCs that is responsive to IL-2 and IL-15 for NK-cell differentiation (25).

Similar to normal CD56^bright NK cells, the majority of cells expanded in such cultures express C-lectin NK receptors (CD94/NKG2), whereas most lack the Ig-superfamily KIR (T.A. Fehniger et al., unpublished observations). The bone marrow–derived CD56^bright NK cells can also express CD2, CD16, and the intracellular zeta (ζ) chain associated with these molecules (18), and can produce large amounts of IFN-γ that are comparable to CD56^bright NK cells found in blood (25). Interestingly, 3–4 weeks are required for the in vitro expansion of CD56^bright NK cells from CD34⁺ HPCs in vitro, similar to the time required for the initiation of human NK-cell expansion in vivo. T cells are not produced under either of these culture conditions (22, 24, 27, 28). Thus, it seems plausible that low-dose IL-2 therapy could serve to selectively enhance the differentiation of human NK cells from bone marrow–derived HPCs, much the same way exogenous administration of G-CSF has been shown to potentiate HPC differentiation along the myeloid lineage (29). It is also possible that the increase in absolute NK-cell number results in part from migration of NK cells from peripheral tissues. However, with the exception of the gravid uterus (30), extravascular tissue sites of CD56^bright NK cells have not been well characterized, leaving this hypothesis difficult to test.

In summary, the selective in vivo expansion of CD56^bright NK cells during prolonged infusion of low-dose IL-2 does not appear to result from enhanced proliferation within this lymphocyte subset. The in vitro and in vivo data provided in this report suggest that this expansion is the result of an IL-2–dependent delay in the onset of programmed cell death in combination with enhanced NK-cell differentiation from bone marrow–derived CD34⁺ HPCs.

The authors thank C. Stewart, R. Baiocchi, and T. Renno for helpful discussions during the course of this study. This work was supported by grants P30CA-16058, CA-68456, and CA-65670 from the National Institutes of Health and by the Coleman Leukemia Research Fund. T.A. Fehniger is the recipient of the Bennett and Medical Scientist Program Fellowships from The Ohio State University College of Medicine.

Todd A. Fehniger and Eric M. Bluman contributed equally to this work.

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