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The Journal of Immunology, 2003, 171: 683-690.
Copyright © 2003 by The American Association of Immunologists

IL-15-Mediated Induction of LFA-1 Is a Late Step Required for Cytotoxic Differentiation of Human NK Cells from CD34+Lin- Bone Marrow Cells1

Isabel Barao*, Dorothy Hudig{dagger} and Joao L. Ascensao2,{ddagger}

Departments of * Veterans Affairs Medical Center and {dagger} Microbiology and Immunology, School of Medicine, University of Nevada, Reno, NV 89509; and {ddagger} Division of Hematology and Oncology, George Washington University, Washington, DC 20037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56+CD3- NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34+Lin- bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Differentiation of mature, cytotoxic NK cells (1) restores critical innate immunity to patients after bone marrow transplantation. After transplantation, production of NK cells takes weeks (2, 3, 4), and faster development of NK cells would benefit patients. Our current scientific understanding of NK cell differentiation is incomplete and puzzling. It is promoted by CD4+ T cell cytokines such as IL-2, but will occur in athymic (5, 6) and IL-2-deficient mice (7). NK cells are a phenotypically distinct population of lymphocytes (CD56+CD3-) (8) that constitutively contain large cytotoxic granules, unlike cytotoxic CD8+ T cells that acquire granules after encounter with Ag. Thus, granules are an indication of NK cell maturation. The granules contain perforin and granzyme B (9, 10), proteins that trigger pathways leading to necrosis (membrane damage) and/or apoptosis (nuclear damage) of target cells (11).

We have found that cells with the CD56+CD3- NK phenotype can be generated in vitro from CD34+Lin- bone marrow (BM)3 cells cultured in the presence of IL-1{alpha}, stem cell factor (SCF), and IL-7 (12); however, these cells lacked cytotoxicity. Other cells, differentiated with IL-2 instead of IL-7, had cytotoxicity as well as the NK phenotype. Both the cytotoxic IL-2-differentiated cells and the noncytotoxic IL-7-differentiated cells had perforin (13). In this study, we show that secondary culture of the IL-7-differentiated cells with IL-2 or IL-15 for only 1 wk will induce cytotoxicity against K562 targets. Thus, after 5 wk of primary culture with IL-7, the cells were still able to acquire the missing component(s) needed for NK lytic activity. The objective of the present study was to determine the cause(s) of the reduced cytotoxicity of the IL-7-differentiated cells and to identify critical changes associated with induction of cytotoxicity. We examined the expression of granzyme B and found little difference between the IL-2- and IL-7-differentiated cells. We next considered other molecules involved in the process of NK killing.

NK cell-mediated cytotoxicity involves target cell binding before release of the cytotoxic granules. Cellular adhesion molecules establish critical binding, termed conjugate formation, between NK cells and target cells (14), and are also implicated in the signals required for granule release. LFA-1 (on lymphocytes) engages ICAM-1 (on targets) as a dominant pathway of adhesion for cytotoxic lymphocytes (15, 16, 17). LFA-1 is a heterodimeric receptor formed by the association of the {alpha}L integrin chain (CD11a) with the {beta}2 integrin chain, CD18 (15). Ab blockade of LFA-1 inhibits NK cell-mediated cytotoxicity (18, 19). Moreover, NK cells from LFA-1-deficient mice are unable to kill target cells (20, 21). Patients with leukocyte adhesion deficiency disease, a genetic defect affecting CD18 (22, 23), lack both LFA-1 and NK activity (24, 25). We turned our attention to the expression of LFA-1 during NK cell differentiation.

The results we present in this work reveal that the IL-7-differentiated CD56+CD3- cells have low expression of LFA-1 and low capacity to form conjugates with K562 targets. LFA-1 can be up-regulated by cytokine-mediated differentiation of the potential killer cells. After 1 wk of secondary culture with IL-2 or IL-15, LFA-1 was expressed by 50% of the cells. The cells with LFA-1 were cytotoxic, while the cells lacking LFA-1 remained noncytotoxic. Abs to LFA-1 blocked the induced lysis. The study provides evidence that the low expression of LFA-1 is a major cause of the poor cytotoxic activity of IL-7-differentiated cells and that LFA-1 expression is an important marker for the development of competent NK cells after bone marrow transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of human BM progenitors

BM was aspirated from the posterior iliac crests of over 20 normal adult volunteers after informed consent was obtained. BM mononuclear cells were isolated by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) centrifugation. CD34+ cells were purified by positive immunomagnetic selection using a CD34 isolation kit, and according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The cells were then sorted in a BD Biosciences FACScan (BD Biosciences, San Jose, CA) using the following mAbs: FITC-conjugated mAbs anti-CD2, anti-CD3, anti-CD4, anti-CD7, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD56 (BD Biosciences), and glycophorin A (Amac, Westbrook, ME), and PE-conjugated mAb to anti-CD34 (BD Biosciences). Reanalysis of the sorted populations was done to ensure the purity. CD56+ cells were not detected in the CD34+Lin- cell populations.

Culture of BM progenitors

Ten thousand CD34+Lin- BM cells were cultured in 0.2 ml of culture medium in 96-well U-bottom plates (Falcon; BD Labware, Mountain View, CA). The medium consisted of IMDM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated human AB serum (North American Biologicals, Miami, FL) and 1% penicillin G sodium (10,000 U/ml)-streptomycin sulfate (10,000 mg/ml) (Life Technologies). The cultures were further supplemented with the following cytokines purchased from PeproTech (Rocky Hill, NJ): 50 ng/ml of SCF (2.7 x 10-9 M final concentration), 100 U/ml of IL-1{alpha} (5.6 x 10-12 M final concentration), and 1,000 U/ml of IL-2 (6.5 x 10-9 M final concentration) or 1,000 U/ml of IL-7 (5.7 x 10-9 M final concentration). The cultures were maintained in a humidified air atmosphere at 37°C and 5% CO2 and fed twice per week by removing 0.1 ml of the supernatant and replacing it with fresh medium containing the cytokines. At week 5, multiple wells were pooled, and the cells were washed thoroughly and analyzed for proliferation, phenotype, cytotoxicity, and intracellular expression of granzyme B. A total of 1,000 U/ml of IL-2 or 500 U/ml of IL-12 (7.1 x 10-10 M final concentration) or 100 U/ml of IL-18 (5.5 x 10-7 M final concentration) purchased from PeproTech, or 100 U/ml of IL-15 (3.8 x 10-9 M final concentration) (a kind gift from Immunex, Seattle, WA, or purchased from PeproTech) was added to the IL-7-initiated cultures, which were then incubated for 1 more wk. After this period of time, cultures were washed thoroughly and cells were collected and analyzed for phenotype, cytotoxicity, and intracellular expression of granzyme B. In one experiment, CD3+ were present in the cultures and were depleted with BioMag magnetic particles covalently attached to an anti-CD3 mAb, according to the manufacturer’s protocol (PerSeptive Diagnostics, Cambridge, MA).

Culture of target cells

K562 targets (a human erythroleukemia cell line) were cultured in 75-cm2 flasks containing IMDM (Life Technologies) supplemented with 10% FCS (Sigma-Aldrich) and 1% penicillin G sodium (10,000 U/ml)-streptomycin sulfate (10,000 mg/ml) (Life Technologies). Cultures were maintained in a humidified atmosphere at 37°C and 5% CO2, and the culture medium was replaced weekly with fresh medium. Before use, these cells were washed twice in medium at room temperature. Viability was assessed by trypan blue exclusion (Life Technologies) and was higher than 95%.

Proliferation of cultured cells

The proliferation index (PI) was calculated by dividing the total number of cells at time of analysis by the number of cells present on day 0 (beginning of culture), as shown in the following equation: PI = (total number of cells at time of analysis)/number of progenitors plated at day 0.

Phenotype of the cultured cells

Phenotypic analysis was performed by flow cytometry using a FACScan flow cytometer (BD Biosciences) and direct three-color staining with the following mAbs from BD Biosciences: FITC-conjugated anti-CD2, anti-CD11a, anti-CD16, anti-CD18, anti-CD56; PE-conjugated mAbs anti-CD56 (Becton Dickinson) and anti-granzyme B (Caltag, Burlingame, CA); and PerCP-conjugated mAb anti-CD3 (BD Biosciences). Controls included FITC-, PE-, and PerCP-conjugated isotype-matched Igs. After washing the cells twice in IMDM supplemented with 5% FCS (HyClone, Logan, UT), they were labeled with a saturating concentration of mAb for 15 min at room temperature in the dark. The cells were then washed twice in PBS-0.5% sodium azide and fixed with 1% paraformaldehyde. At least 10,000 cells per aliquot were analyzed and gated on the presumptive lymphocyte region, as defined by forward and side scatter. Upon analysis, quadrants were positioned to allow at least 99% of the control, isotype-exposed population to remain in the negative quadrant. NK cells were phenotypically defined as CD56+CD3-.

Cytotoxicity assays

Cells were tested for cytotoxicity against K562 cells in a standard 4-h 51Cr release assay. A total of 1 x 106 target cells was washed and incubated for 90 min at 37°C with Na251CrO4 (NEN Life Science, Pittsburgh, PA) at 0.1 mCi/106 target cells. The cells were washed five times in IMDM supplemented with 5% FBS and counted. Effector cells harvested from the cultures on the day of analysis were washed, counted, assessed for their viability by trypan blue exclusion, and placed at 5 x 103 cells/well in V-shaped microwell plates (Applied Scientific, South San Francisco, CA) at E:T ratios that ranged from 20:1 to 2.5:1. The plates were then centrifuged at 120 x g for 3 min and incubated for 4 h at 37°C in a 5% CO2 humidified air atmosphere. After this period, the plates were centrifuged at 200 x g, and 0.1 ml of the supernatants was removed from each well and withdrawn into aliquots of 1 ml of liquid scintillation cocktail (Wallac, Gaithersburg, MD). Radioactivity was measured in a scintillation counter 1450 Microbeta (Wallac). All determinations were done in triplicate, and percentage of lysis was determined using the following equation: percentage of specific lysis = (experimental mean cpm - spontaneous release mean cpm)/(total release mean cpm - spontaneous release mean cpm) x 100. Total 51Cr release was determined by adding 0.1 ml of 1% SDS solution (Sigma-Aldrich) to labeled target cells. Spontaneous 51Cr release, as determined by adding 0.1 ml of supplemented medium to target cells, averaged 15%. Cytotoxicity assays were also performed on LFA-1-negative and LFA-1-positive NK cell subsets after these were isolated by sorting using FITC-conjugated mAbs anti-CD11a and anti-CD18 (BD Biosciences); Per-CP-conjugated mAb anti-CD3 (BD Biosciences); and PC-5-conjugated mAb anti-CD56 (Beckman Coulter, Brea, CA). Controls included FITC-, PE-, and PC-5-conjugated isotype-matched Igs.

Blocking Ab assays

A total of 5 x 105 cells was treated with goat serum for 30 min at room temperature to prevent nonspecific labeling. Cells were then washed twice in PBS and incubated for 45 min at 4°C with a combination of anti-CD11a (Endogen, Woburn, MA) and anti-CD18 mAb (BD Biosciences) at a concentration of 5 µg/ml each. Cytotoxicity assays were next performed against K562 targets, as described above. Controls included NK cells incubated with 10 µg/ml of an isotype-matched, irrelevant mouse IgG1 control. The mAbs remained present during the assay.

Effector-target conjugate formation

NK cells were stained with FITC-conjugated mAb anti-CD56 (green fluorescence) from BD Biosciences for 15 min at room temperature in the dark and washed twice in PBS. K562 targets were labeled intracellularly with 253 µM hydroethidine (Polysciences, Warrington, PA) for 30 min at 37°C (red fluorescence) and washed twice in PBS. The effectors and targets (105 cells/ml of each) were mixed together, pelleted, and incubated at 37°C for 15 min. The pellet was gently resuspended and transferred to 1 ml of ice-cold IMDM medium (Life Technologies). Conjugate formation (simultaneous green and red fluorescence) was quantified using FACScan (BD Biosciences). At least 10,000 lymphocytes were analyzed per aliquot. The percentage of binding was calculated as follows: percentage of conjugates formed = (number of NK cells bound to target cells)/(total number of NK cells) x 100. The variability of conjugate formation was ±5% among different experiments. Adhesion assays were performed on LFA-1-negative and LFA-1-positive NK cell subsets after these were isolated by sorting using FITC-conjugated anti-CD11a and anti-CD18 mAbs (BD Biosciences); Per-CP-conjugated mAb anti-CD3 (BD Biosciences); and PC-5-conjugated mAb anti-CD56 (Beckman Coulter), as described above. For adhesion inhibition studies, anti-CD56-stained cells were incubated with purified anti-CD11a and anti-CD18 mAbs or with irrelevant mouse IgG1 control (negative binding control), as described above.

Data presentation and statistical analysis

The data are presented as the mean ± SD of the results from multiple experiments. A paired Student’s t test was used to determine the statistical significance of differences in the data. Differences were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-7-differentiated cells are noncytotoxic, but have granzyme B

CD34+Lin- bone marrow cells were cultured for 5 wk with SCF, IL-1{alpha}, and IL-7. An average of 26.7% of the cells differentiated into cells expressing the NK marker CD56 (Table I) (4). They had only 2.6% cytotoxicity at the E:T ratio of 20:1, while IL-2-cultured cells had >63.2% cytotoxicity at the same ratio (not illustrated). The difference in cytotoxicity between the IL-7 and IL-2 cultures was statistically significant (n = 5; p < 0.001) and has been previously reported (12). There were fewer CD56+ cells produced with IL-7, 26.7% compared with the 85.4% produced with IL-2 (Table I), but the ~3-fold lower frequency of potential effector cells was insufficient to explain the absence of cytotoxicity.4 Both the IL-7- and IL-2-generated CD56+CD3- cells had substantial levels of granzyme B (IL-7, Fig. 1A; IL-2, Fig. 1B). The IL-7-cultured cells shown in Fig. 1 were from the same donor as the IL-2-cultured cells and represent cells cultured within one experiment. The mean fluorescent indexes for granzyme B, in an average of five experiments, were less than 2-fold greater for the IL-2-cultured cells, 143.4 ± 49.8 vs 84.0 ± 34.2, respectively, for IL-2- and IL-7-cultured cells. This difference was not statistically significant (p = 0.059) and suggests that something other than the cytotoxic granule protease granzyme B was limiting the cytotoxicity of the IL-7-cultured cells. Previously, we found that both IL-7- and IL-2-cultured cells contained perforin (13), so that it would appear that these cells might be armed, but ineffective as killers. We monitored our K562 cell line for Fas expression and found that it was Fas negative (data not shown), indicating the importance of granule exocytosis mechanism in the experiments reported in this work.


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  		Table I. Phenotype of cells derived from CD34+ Lin- BM cells cultured with IL-2 or IL-7a

 


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  		FIGURE 1. Noncytotoxic cells differentiated with IL-7 contain granzyme B in amounts similar to cytotoxic cells differentiated with IL-2 or IL-15. Intracellular granzyme B was labeled with PE-conjugated mAb anti-granzyme B and analyzed by flow cytometry. The cells displayed were positive for CD56 (FITC-conjugated mAb anti-CD56) and negative for CD3 (PerCP-conjugated anti-CD3). The data displayed are representative of five experiments. A, Cells cultured for 5 wk with IL-7. B, Cells cultured for 5 wk with IL-2. C, Cells cultured for 5 wk with IL-7 were recultured for 1 more wk with IL-2. D, Cells cultured for 5 wk with IL-7 were recultured for 1 more wk with IL-15.

 
Differences in the total number of cell divisions could have left the cells at different stages of cytotoxic maturation. However, the total number of cell divisions supported by IL-7 and IL-2 appears similar. After 5 wk, the proliferation indexes of cells exposed to IL-7 averaged 10.2 ± 2.7, while the indexes averaged 8.8 ± 1.4 for cells exposed to IL-2. These indexes lacked statistical significant difference and indicate that both cultures underwent similar and limited expansion. Thus, the increased frequency of the NK phenotype after IL-2 treatment (presented in Table I) accounts for the numerical increase in NK cells rather than a gross expansion of all cells in response to IL-2. This result contrasts with another study in which IL-2 was used with stromal feeder cells and gross cell expansion and differentiation occurred (26). Furthermore, the stroma-free cells we cultured with IL-7 for 3 more wk (8 wk total) developed more CD56+ cells than were present at 5 wk (to ~50% of the cultures), but the prolonged culture failed to enhance cytotoxicity (E:T, 20:1; K562 lysis averaged 1.8%; data not illustrated) or increase expression of granzyme B.

Lack of LFA-1 on IL-7-differentiated cells

We monitored the levels of several other cell surface markers involved in NK cell cytotoxicity of the CD56+ cells (Table I). The expression of LFA-1 was different, considerably lower for IL-7-differentiated CD56+ cells when compared with IL-2-differentiated CD56+ cells (for CD11a, 4.8 vs 38.3%, p < 0.001; for CD18, 6.7 vs 46.8%, p < 0.001). The expression of two other NK cell surface molecules was depressed regardless of the cytokine. The CD56+ cells had low levels of the adhesion molecule CD2, 2.2 and 4.1%, for IL-7- and IL-2-differentiated cells, respectively. Expression of CD16 (Fc{gamma}RIII), the receptor that mediates Ab-dependent cell cytotoxicity, was also low for both cultures with 6 and 5% for IL-7- and IL-2-differentiated cells, respectively. For reference, mature peripheral blood NK cells have substantial levels of CD2 and CD16 (8, 27, 28). Because both IL-2- and IL-7-cultured cells lacked CD2 and CD16 and only the IL-2-cultured cells were cytotoxic, it would appear that neither of these two markers contributed to the critical differences in cytotoxicity.

Secondary culture of IL-7-differentiated cells with IL-2 or IL-15 up-regulates the expression of LFA-1 and augments their cytotoxic activity

We used secondary cultures to identify the component(s) that the NK-like cells differentiated with IL-7 need to acquire to become cytotoxic. We recultured the IL-7-differentiated cells with individual cytokines rather than with stromal feeder cells, because we hoped to limit the number of potential changes. Given that IL-2 (12, 29, 30), IL-12 (31, 32), IL-15 (33, 34), and IL-18 (35) enhance the cytotoxicity of mature NK cells, we examined the effects of these cytokines on the phenotype and cytotoxic activity of IL-7-differentiated CD56+ cells. At the fifth week of culture, IL-2, IL-12, IL-15, or IL-18 was added separately to the IL-7-induced cultures that were then recultured for 1 more wk.

Phenotypic analyses of the cultured cells are provided in Table II. The percentage of CD56+ cells significantly increased with IL-2 or IL-15, from 26.7 to 38.4% with IL-2 (p = 0.038) and from 26.7 to 42.6% with IL-15 (p = 0.039). Thus, IL-15 was as effective as IL-2. Significant LFA-1 up-regulation also occurred. The expression of the CD11a subunit was increased from 4.8% total positive cells after culture with IL-7 to 19.8% cells positive after they were recultured with IL-2 (p = 0.001). The expression of the CD11a subunit was increased to a similar extent, 21.5% total cells positive after reculture with IL-15 (p < 0.001). Induction of the CD18 subunit was comparable to the induction of CD11a. CD18 was expressed on 6.7% of the total cells cultured with IL-7 and increased to 28.7% of the cells cultured with IL-2 (p < 0.001) and to 30.4% with IL-15 (p < 0.001). It should be noted that 1 wk of reculture with IL-2 was nearly as effective as 5 wk of continuous exposure to IL-2 for induction of LFA-1 cells (~40% cells positive at 5 wk; Table I). Fig. 2 illustrates the induction of LFA-1 on the CD56+ cell population. About one-half of the CD56+CD3- cells became positive for either LFA-1 subunits, and IL-15 (Fig. 2, E and F) was as effective as IL-2 (Fig. 2, C and D). Cells cultured with IL-15 induced CD11a without concurrent induction of CD11b. This observation guided our later selection of a combination of anti-CD11a and anti-CD18 Abs to optimally block LFA-1. In contrast to the changes in LFA-1, the percentages of expression of granzyme B were unaltered after IL-7 cells were recultured with IL-2 or IL-15 (Fig. 1, C and D).


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  		Table II. Phenotype of IL-7-differentiated cells stimulated with IL-2, IL-12, IL-15, or IL-18a

 


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  		FIGURE 2. Reculture of IL-7-differentiated cells with IL-2 or IL-15 up-regulated expression of LFA-1. LFA-1 was monitored with fluorescent mAbs to the CD11a and CD18 subunits. The cells displayed were positive for CD56 (PE-conjugated mAb anti-CD56) and negative for CD3 (PerCP-conjugated anti-CD3), and the percentages indicated on each figure represent the fraction that was positive for either subunit. The data displayed are representative of five experiments. A and B, Cells cultured with IL-7 alone had undetectable levels of CD11a (A) and CD18 (B) compared with the isotype controls used to set the gates. C and D, About one-half of the cells recultured for 1 wk with IL-2 had substantial levels of CD11a (C) and CD18 (D). E and F, A similar fraction, about one-half, of the cells recultured for 1 wk with IL-15 had substantial levels of CD11a (E) and CD18 (F).

 
In parallel with these phenotypic changes, IL-2- or IL-15-differentiated cells acquired cytotoxic activity. At the E:T ratio of 20:1, IL-2 and IL-15 increased lysis from less than 3% to 34.9 (p < 0.001) and 38.8% (p < 0.001), respectively (Fig. 3). The increased lytic activity was well over 8-fold when evaluated on the basis of lytic units. The activity for E:T ratio of 2.5:1 for the IL-2- and IL-15-treated cells exceeded the activity of the E:T of 20:1 of the control (IL-7-cultured) cells. The NK cytotoxicity increased much more than the increased frequency of CD56+ cells after reculture with the cytokines, 8-fold (Fig. 3) compared with ~2-fold (Table II), respectively. Neither IL-12 nor IL-18 affected the NK phenotype (Table II) or lysis (Fig. 3).



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  		FIGURE 3. Cells recultured with IL-2 or IL-15 acquired cytotoxic activity. Cells were recultured with IL-2, IL-12, IL-15, or IL-18 for 1 wk and then assayed at the indicated E:T ratios with 51Cr-labeled K562 cells as targets. The percentages of lysis represent the mean ± SD for five experiments. IL-2 and IL-15 induced significant cytotoxicity compared with IL-7-cultured cells at all the E:T ratios indicated (p < 0.05). In contrast, cells recultured with IL-12 or IL-18 remained as nontoxic as the cells cultured only with IL-7.

 
Role of LFA-1 in the cytotoxicity of IL-2- and IL-15-differentiated cells

To evaluate the contribution of the newly expressed LFA-1 to cytotoxicity, we incubated the cells with anti-LFA-1 mAbs CD11a and CD18 and then assayed lytic activity. The mAbs significantly reduced the cytotoxic activity (Fig. 4). The activity of the IL-2 secondarily differentiated cells decreased from 29.6 to 17.9% at the E:T of 20:1 (Fig. 4A, p = 0.003). The cytotoxicity of cells stimulated with IL-15 was similarly diminished, from 35.5 to 21.5% at the E:T ratio of 20:1 (p = 0.005), reducing it to the lysis of the control cells at the E:T of 2.5:1 (20%, Fig. 4B). In both A and B of Fig. 4, there was an ~8-fold loss of activity by lytic units. This inhibition is of comparable magnitude with studies that applied anti-LFA-1 Abs to block the lysis mediated by mature peripheral blood NK cells (18, 19).



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  		FIGURE 4. Abs to LFA-1 inhibited lysis by cells recultured with IL-2 or IL-15. The IL-7-cultured cells were recultured with either IL-2 or IL-15 for 1 wk, harvested, preincubated with a combination of 5 µg/ml of each mAb to the CD11a and CD18 subunits of LFA-1 for 45 min, and then assayed with 5 µg/ml of each of the mAbs at the indicated E:T ratios. The controls received either no Ab or 10 µg/ml of isotype-matched, control Ig. The percentages of lysis represent the mean ± SD for five experiments. K562 lysis increased with increased E:T ratios. The Abs decreased lysis by ~8-fold, reducing the 20:1 E:T lysis with Abs to the lysis mediated by the 2.5:1 E:T controls without Ab. A, Cells recultured with 1000 U/ml IL-2. B, Cells recultured with 100 U/ml IL-15.

 
As further indication of the importance of the acquisition of LFA-1, cells were sorted for the CD56+CD3- cells that were either positive or negative for CD11a and CD18 Ag expression, and then tested for cytolytic activity. The LFA-1+ cells were cytotoxic, and the LFA-1- cells were not cytotoxic (Fig. 5). At an E:T ratio of 20:1, there was 30% lysis by the LFA-1+ subset and only 5.7% of K562 lysis by the LFA-1- subset from the secondary IL-2-differentiated cells (Fig. 5A). Results were very similar for the secondary IL-15-differentiated cells (Fig. 5B).



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  		FIGURE 5. The cells that acquired LFA-1 after reculture with IL-2 or IL-15 were the cells that also acquired lytic activity. Cells that had been recultured with either IL-2 or IL-15 for 1 wk were sorted for CD56+CD3- cells that differed in their expression of LFA-1. Next, the sorted cell populations were assayed for lysis of K562 cells. The LFA-1-negative cells had little lytic activity even at 20:1 E:T ratios. The percentages of lysis represent the mean ± SD for five experiments. A, Cells recultured with 1000 U/ml IL-2. B, Cells recultured with 100 U/ml IL-15.

 
LFA-1 supports adhesion of cultured cells to K562 targets

IL-7-differentiated cells exhibited low conjugate formation with K562 cells (1.8% of the CD56+ cells were in conjugates; Fig. 6A). Secondary reculture with IL-2 or IL-15 induced a significant increase in the formation of CD56+ conjugates to 14.8 (p = 0.008) and 11.7% (p = 0.004), respectively (Fig. 6A). The frequency of conjugate-forming CD56+CD3- cells was lower than what one might expect from cells that were 50% LFA-1 positive. However, for reference, using the same flow cytometric adhesion assay, the frequency of peripheral blood CD56+CD3- cells that formed conjugates was similar, and shear forces may have disrupted some conjugates (data not shown).



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  		FIGURE 6. Cells recultured with IL-2 or IL-15 acquired the capacity to bind targets, and the capacity was dependent upon LFA-1. Binding was measured as conjugate formation between the cultured cells and K562 target cells, using a flow cytometric assay. In the assay, the cultured cells were identified by green fluorescence from FITC-conjugated anti-CD56 mAb, and the target cells were identified by red fluorescence from hydroethidine treatment. Cultured cells and target cells were mixed at a 1:1 ratio. Conjugates were detected as cytometric events with double (red and green) fluorescence. A, Induction of conjugate formation after reculture with either IL-2 or IL-15. Values represent the means ± SD for three experiments. IL-2 or IL-15 was equally able to induce conjugate formation, p < 0.05, compared with the IL-7-cultured cells. B and C, Binding capacity was dependent upon LFA-1. The cytokine used for culture is marked on the figures. After culture, the cells were preincubated with a combination of 5 µg/ml of mAbs to each of the LFA-1 subunits (CD11a and CD18) for 45 min and then assayed for conjugate formation in the presence of both mAbs. Controls were incubated with either no Abs or 10 µg/ml of the isotype, control Ig. Values represent the mean ± SD for three experiments. Anti-LFA-1 Abs significantly inhibited conjugate formation for cells cultured with either cytokine, p < 0.05.

 
The role of LFA-1 in the conjugates was evaluated by Ab-inhibition experiments. Adhesion assays were performed in the presence of LFA-1-blocking mAbs anti-CD11a and anti-CD18. Ab to LFA-1 diminished the capability of the cells to form conjugates (from 14.8 to 5.8%, p = 0.035; and from 11.7 to 4.1%, p = 0.009, for cells restimulated with IL-2 or with IL-15; Fig. 6, B and C, respectively). Some conjugate-forming cells persisted in the presence of the Abs. No significant effect was noted upon incubation with irrelevant mouse IgG1.

Adhesion assays were also performed on IL-2- or IL-15-treated cell populations sorted for the expression of CD11a and CD18 Ags. Surprisingly, the percentage of conjugates formed between K562 target cells and LFA-1-negative or LFA-1-positive cell subsets was comparable (not illustrated). A maximum of 10.4% of conjugate formation was observed by the LFA-1-negative cell subset. These LFA-1-negative cells are about one-half the CD56+CD3- cell population after IL-2 or IL-15 cytokine differentiation. In aggregate, the data are consistent with the possibility that for LFA-1+ cells, LFA-1 is important for conjugate formation, while for cells that lack LFA-1 other adhesion or receptor molecules may support conjugate formation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study extends our knowledge of the effects of different cytokines on the differentiation of NK cells from bone marrow progenitor cells. IL-7 was sufficient to induce granzyme B as well as perforin, both hallmark proteins of the granules of cytotoxic lymphocytes. However, these components that enable death by cytotoxic granule exocytosis were insufficient to confer cytotoxic activity to the cells. Thus, the immature NK cells may have had granules, but have been unable to deliver them. In contrast, the cytokines IL-2 or IL-15 completed the cytotoxic maturation of the NK cells. The cells still lacked CD2 and CD16, and therefore had acquired cytotoxicity, but lacked the mature NK cell phenotype typical of peripheral blood NK cells. The IL-2 or IL-15 cytotoxic maturation was accompanied by de novo expression of LFA-1. Furthermore, the LFA-1 on these cells participated in their cytotoxic activity.

The ability of IL-7 to support differentiation of CD56+CD3- cells and formation of two cytotoxic granule proteins (granzyme B and perforin) from the CD34+Lin- bone marrow progenitors is noteworthy. IL-7 is unnecessary for NK cell development in vivo: IL-7-deficient mice lack T and B cells, but have mature NK cells with lytic activity (36, 37, 38). Our data suggest that IL-7 may be able to support formation of functional cytotoxic granules, but that exocytosis of these granules requires induction of other NK cell proteins.

It has been known for several years that IL-15 would support NK cell generation (39). Mice deficient in IL-15 or the {alpha}-chain of the IL-15R (knockout mice) lack mature NK cells (40, 41), indicating that IL-2 alone is insufficient. It had been thought that IL-15 was needed primarily as a growth factor for the NK cells differentiated in the bone marrow (34, 38). However, in our experiments, IL-7, without IL-15 or IL-2, supported growth of cells with a CD56+ NK phenotype. The IL-15-mediated induction of LFA-1 in NK progenitor cells is a new function for IL-15. It has also been recently reported that IL-15 treatment can up-regulate LFA-1 and CD2 on PBMCs (42, 43). A major difference between these experiments and the ones reported in this study is that the circulating blood NK cells were fully differentiated and already had both LFA-1 and CD2 adhesion proteins. In our experiments, we started with Lin- bone marrow cells. Our critical new observations are that IL-15 induced LFA-1 in NK progenitors that previously lacked both LFA-1 and CD2, and that IL-15 did so without also simultaneously inducing CD2. It can be concluded that IL-15 has a distinct and more specific effect on NK immature cells.

It should be noted that, even though IL-15 appears physiologically essential for NK cell development and is important for events that occur late in maturation, IL-15 alone is insufficient to induce the complete phenotype characteristic of mature NK cells found in peripheral blood. For example, Miller and McCullar (44) found that direct contact with stromal cells was required for induction of the killer Ig-like receptors CD158a and CD158b, and that their cytokine mix (which included 10 ng/ml of IL-15) was insufficient without stromal cells. Briard et al. (45) also found that direct contact with stromal cells (which produced IL-15) was necessary for induction of the NK receptors CD94, and p30, p44, and p46, but under their conditions the NK-like cells still lacked CD158a and CD158b. Our LFA-1-positive, lytic cells also lacked CD158a and CD158b (data not shown). These two killer Ig-like receptors help mediate the exquisite discrimination of self from nonself. Thus, the requirements for final induction to achieve the complete human NK phenotype remain a challenging goal for future research.

The critical role of IL-15 induction of LFA-1 is supported by several observations. Anti-LFA-1 Abs blocked both binding and killing mediated by IL-15-differentiated cells. The K562 cells express the counterligand CD54 specifically recognized by LFA-1 (14, 46). The CD18 subunit of LFA-1 is a signaling molecule (47, 48), and engagement of LFA-1 promotes cytotoxic granule exocytosis from NK cells (42). Thus, both the LFA-1 ligand and its counterligand are present, and the consequences of their interaction directly impact the granule-dependent cytotoxicity required for K562 lysis. Furthermore, NK activity toward many targets other than K562 is supported by LFA-1 interactions, including MOLT-4, HSB-2, Jurkat (49), CEM (50), and the rodent NK target YAC-1 (51). However, for perspective, readers are advised that some NK cell-target cell interactions are lytic even when CD11a and CD18 are blocked (e.g., the NK 3.3 cell line still killed MOLT-4) (49).

Both IL-2 and IL-15 supported LFA-1 induction and NK differentiation at ~10-9 M concentrations of the cytokines in vitro. An issue is whether both cytokines act physiologically on bone marrow progenitors or whether only IL-15 has a physiological role. In vivo, IL-2 fails to replace IL-15 for NK cell differentiation (40, 41). IL-15 knockout mice can make IL-2, but lack circulating NK cells. In the bone marrow, stromal cells normally secrete IL-15, but not IL-2 (34). How can we explain the effects of IL-2 in our in vitro system? Perhaps the effects depend on the ability of IL-2 to bind to the IL-15R. IL-2R and IL-15R share {beta}- and {gamma}-chains (52, 53), and this dimer can bind IL-2 with moderate affinity (Ka = 109 M-1). The dimeric receptor would be occupied by 6 x 10-9 M IL-2 used in our experiments. (IL-15 was in ~100-fold excess of concentrations needed to activate the trimeric IL-15R (Ka = 1011 M-1) and requires the IL-15 {alpha}-chain for activity.) The IL-7-differentiated cells lack the IL-2R {alpha}-chain, CD25 (data not shown). Thus, in vitro, high concentrations of IL-2 can support differentiation of bone marrow cells, but in vivo only IL-15 may be able to perform this role. This scenario relates to bone marrow transplantation because NK cells appear before other components of the immune system and IL-2 is produced by the CD4+ T cell component.

A therapeutic role for IL-15 may be very important. Administration of IL-2 to patients has severe and potentially life-threatening side effects (54), while IL-15 may have less serious side effects (55, 56). The ability of IL-15 to induce LFA-1 within 1 wk of treatment suggests that short cytokine therapy might suffice to promote cytotoxic differentiation of NK cells after bone marrow transplantation.

IL-12 and IL-18 lacked effects on the IL-7-differentiated cells even though the cytokines were at concentrations sufficient to engage their respective receptors (57, 58) and even though these cytokines increase the cytotoxic activity of mature peripheral blood NK cells (31, 32, 35). We interpret these results as additional information that the IL-7-cultured cells in our system are at an intermediate stage of differentiation, containing perforin and granzyme B, but nonlytic and lacking CD2 and CD16.

This work documents a new role for IL-15. It is critical for LFA-1 induction in NK cells. Administration of IL-15 might promote the development of innate immunity after bone marrow transplantation. Monitoring the levels of LFA-1 may also be important to identify functionally mature NK cells after bone marrow transplantation.


   Acknowledgments
 
We thank Dr. Elaine Thomas of Immunex for generously providing human rIL-15.


   Footnotes
 
1 This research was supported in part by a scholarship from the Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) and the following grants: National Institutes of Health R01CA38942 (to D.H.) and Veterans Administration Research (to J.L.A.). Back

2 Address correspondence and reprint requests to Dr. Joao Ascensao, 2150 Pennsylvania Avenue, NW, 3-428, Washington, DC 20037. E-mail address: jascensao{at}mfa.gwu.edu Back

3 Abbreviations used in this paper: BM, bone marrow; SCF, stem cell factor. Back

4 For the record, it should be noted that outgrowth of competing T and B cells was lacking in the cultures with IL-7 and will not explain the lower frequency of CD56+ cells. T cells had a maximum of 8.4% of CD3 expression, and B cells (CD19+ or CD20+) were undetectable. Back

Received for publication January 28, 2003. Accepted for publication May 6, 2003.


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