Fibroblasts from Human Spleen Regulate NK Cell Differentiation from Blood CD34⁺ Progenitors Via Cell Surface IL-15

Isolation of fibroblasts from human spleen tissue

Normal adult spleen samples (n = 4) were obtained from both male and female donors, aged 49–70 years, from whom consent for multiple organ donation had been granted (Etablissement Français des Greffes, Kremlin-Bicêtre, France). Fibroblasts were isolated by gentle grinding of splenic tissue in a sterile glass homogenizer. Tissue homogenates were then centrifuged at 1000 rpm for 5 min. Cellular aggregates present in the pellet were washed three times with PBS and then transferred to culture flasks in a small volume of RPMI 1640 medium (Life Technologies, Cergy-Pontoise, France) supplemented with 15% FCS (Life Technologies) to adhere. The medium was changed every 3–4 days until a substantial outgrowth of cells was observed, at which point cells were trypsinized (Life Technologies) and subsequently subcultured at a 1:2 split ratio for up to three passages before analysis. The fibroblastic nature of the cultures was assessed by their homogeneous staining for anti-smooth muscle-actin with a typical stress fiber distribution characteristic of myofibroblasts (8). All cells also showed strong staining for other mesenchymal Ags, including vimentin, fibronectin, and collagen type I and III. Homogeneous cell surface expression of Ag ASO2, specific for fibroblasts (9), was also detected by flow cytometry, whereas Ag CD31, characteristic of endothelial cells, was missing.

Isolation of human CD34⁺ cells from adult resting peripheral blood

Mononuclear cells obtained from the peripheral blood of healthy donors (n = 9) were prepared by centrifugation over a Ficoll solution (specific gravity, 1.077; Pharmacia Biotech, Orsay, France). After overnight plastic adherence in culture medium, the nonadherent mononuclear CD34⁺ cell fraction was isolated using a supermagnetic microbead selection kit and varioMACS columns according to the manufacturer’s instructions (Miltenyi Biotec, Paris, France). Purity was >90%.

Culture of CD34⁺ cells with human spleen-derived fibroblasts

Fibroblasts were grown to confluence in 60-mm petri dishes (Falcon; BD Biosciences, Le Pont de Claix, France) or six-well and 0.4-μm pore size Transwell plates (PolyLabo, Strasbourg, France) and incubated for 3 wk with CD34⁺ cells (3:10 ratio with fibroblasts) in standard culture medium supplemented with 10% FCS; no exogenous cytokines were added. Half-medium changes were performed once a week. In some experiments cultures were also treated with various combinations of neutralizing Abs as follows. Anti-stem cell factor (anti-SCF³; 1 μg/ml) goat polyclonal Ab, 60 ng/ml anti-Flt3 ligand (anti-Flt3L) mAb 40416.111, and 100 ng/ml anti-IL-15 mAb M111 (R&D Systems, Abingdon, U.K.) were added at the start of experiments and subsequently at each medium change. At the indicated times nonadherent cells were harvested by washing and counted, and cell viability was determined by the trypan blue dye exclusion test. Analysis of monolayer-derived fibroblasts was performed after a 3-min trypsinization at 37°C.

Cell immunophenotyping

Cells were stained with various combinations of mAbs as listed in Table I⇓ according to conventional direct and indirect techniques (6). In some experiments cell membranes were permeabilized by treatment with ORTHOPermeafix (Ortho Diagnostic Systems, Roissy, France) before Ab incubation for monitoring total (surface and cytoplasmic) Ag expression. Unspecific staining was determined using identical nonreactive isotype mAbs. Cells were analyzed on a FACScan (BD Biosciences, Franklin Lakes, NJ) using CellQuest (BD Biosciences) and WinMDI software programs (Scripps Research Institute, La Jolla, CA).

Cytokine production

Multiple aliquots of cell-free supernatants were collected from fibroblasts cultured alone or with hemopoietic CD34⁺ cells, filtered, and frozen at −20°C. Standard ELISA for IL-2, IL-15, SCF, Flt3L, TNF-α, and IFN-γ were performed using commercial kits from R&D Systems according to the manufacturer’s instructions.

Confocal microscopic analysis

For double staining, cells were first permeabilized with ORTHOPermeafix for 45 min at room temperature. Cells were then stained with anti-perforin mAb δG9 (BD PharMingen, Le Pont de Claix, France), followed by incubation with Alexa Fluor 594-conjugated goat anti-mouse Ab (Molecular Probes, Montluçon, France). Cells were then labeled with biotinylated anti-granzyme B mAb CLB-GB11 (Tebu, Le Perray en Yvelines, France), followed by streptavidin-Alexa Fluor 488 conjugate (Molecular Probes). Cytospin-stained cells were analyzed by laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany).

Cytotoxicity assay

The cytolytic activity of total nonadherent cultured cells was determined in a standard 4-h ⁵¹Cr release assay against the NK-sensitive K562 cell line, a gift from Dr. S. Chouaib (IGR, Villejuif, France). In some experiments cells were additionally incubated with 1000 U recombinant human IL-2 (Hoechst-Marion-Roussel, Romainville, France) for 48 h and then tested for cytotoxic activity.

Characteristics of CD34⁺ progenitors isolated from resting blood

The purified CD34⁺ cell populations isolated from adult nonmobilized peripheral blood cells showed no evidence of myeloid or NK and B lymphoid differentiated cells, with generally <1% of the population expressing CD14, CD15, CD56, and CD19 (data not shown). In contrast, cells expressing lymphoid CD2 (19.0 ± 9.6%; range, 3–28%) and megakaryocyte CD41 (46.4 ± 26.9%; range, 22–89%) lineage cell Ags were detected, indicating that CD34⁺ progenitors in resting blood were most likely committed cells (data not shown). Further phenotype characterization also indicated that significant (44–76%) proportions of CD34⁺ cells expressed a set of surface adhesion molecules necessary for blood cell interactions, including ICAM-1, VLA-4, and LFA-1. Cell surface expression of c-kit (23%) and Flt3 (12%) was detected in a fraction of cells (data not shown).

Differentiation of resting blood CD34⁺ progenitors cultured with human spleen-derived fibroblasts

When purified CD34⁺ cells were cultured with splenic fibroblasts for 3 wk, a decline in nonadherent cell number was generally observed by wk 1, followed by a low cell expansion (Table II⇓). Phenotype characterization of nonadherent surviving cells revealed a progressive lost of cells positive for CD34, CD41, and CD2. In contrast, increasing percentages of CD56⁺ and, to a much lesser extent, CD15⁺ cells were detected. No cells positive for CD3, CD19, and CD14 could be identified, indicating the lack of T or B cell and monocyte development (data not shown). However, a fraction (∼40%) of the nonadherent cell population remained consistently negative for each of the Ags tested, indicating that other lineages might also be present.

Phenotype and lytic activity of CD56⁺ cells produced with splenic fibroblasts

The production of CD56⁺ cells that was reproducibly obtained in a series of experiments using fibroblasts and blood CD34⁺ cells from different donors generally reached maximal values of 33.5 ± 6.4% by wk 3 (Fig. 1⇓a). Further analysis demonstrated that these CD56⁺ cells displayed a typical NK phenotype that closely resembled cells produced in stroma-free medium supplemented with IL-15, Flt3L, and c-kit ligand (c-kitL) (14, 15, 16). Thus, dual flow cytometry showed that all CD56⁺ cells lacked CD3, and only a small fraction of them expressed CD2 (<5%) or CD16 (∼10%; Fig. 1⇓b). Furthermore, a high (>80%) percentage of CD56⁺ cells coexpressed the C-type lectin receptor CD94 and the NK receptors p46 (>70%), p44 (>60%), or p30 (∼50%). The surface expression levels of these NK Ags were consistently high (mean fluorescent intensity between logs 2 and 3) in a majority of cells (Fig. 1⇓c). Other specific killer cell IgG-like receptors, including p58-1, p58-2, p70, and leukocyte Ig-like receptor-1, were lacking or expressed in only a small (<5%) percentage of cells (data not shown). Further confocal microcopic examination of NK cells demonstrated in a high proportion of nonadherent cells (Fig. 2⇓) an intracytoplasmic colocalization of perforin and granzyme B, which are thought to be lytic mediators for lymphocyte cytotoxicity (17). Expression of Fas ligand, another mediator responsible for the lytic NK cell function (18), was not detected by flow cytometry (data not shown). When total nonadherent cells were directly tested for their ability to lyse the classical class I-negative target K562, a substantial (>35%) level of lytic activity was detected at an E:T cell ratio of 5:1 (Fig. 1⇓d). Further stimulation by IL-2 did not increase cell cytotoxicity.

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FIGURE 1.

Characterization of NK cells generated with human spleen-derived fibroblasts. a, Kinetics of NK cell development monitored by the percentage of nonadherent CD56⁺ cells harvested at the indicated times. The results represent the mean ± SEM of five separate experiments. b, Percentage of CD56⁺ cells double-stained with the indicated labeled mAbs and analyzed by flow cytometry. c, CD56 and NKR expression levels. d, Percentage of cell lysis with unstimulated (▪) and IL-2-stimulated (□) nonadherent cells at a 5:1 E:T cell ratio. For b–d, results are representative of at least three separate experiments

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FIGURE 2.

Confocal microscopic analysis of intracytoplasmic localization of perforin and granzyme B in NK cells generated from blood CD34⁺ cells cocultured with splenic fibroblasts. a, Background staining. b, The yellow staining indicates intracellular colocalization of perforin (red) and granzyme B (green).

Expression of c-kitL, Flt-3L, and IL-15 in human spleen-derived fibroblasts

Low amounts of the two ligands c-kitL/SCF and Flt3L, related to NK progenitor amplification and differentiation (14, 15), were detected in culture supernatants of spleen-derived fibroblasts as early as day 4, with optimal values of 93.9 ± 24.3 and 19.0 ± 5.2 pg/ml, respectively, by wk 1 (Fig. 3⇓). In contrast, IL-15, the most potent cytokine for NK cell differentiation (14), was barely detectable, below or at the limit of ELISA detection and accuracy (3.9 pg/ml) during the first 4 days of culture and then reached an optimal value of 17.7 ± 0.4 pg/ml by wk 1. Other cytokines, including IL-2, TNF-α, and IFN-γ, consistently remained undetectable (data not shown). Except for SCF, no significant changes in secreted cytokines were observed under coculture conditions.

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FIGURE 3.

Cytokine production in human spleen-derived fibroblasts. Supernatants from splenic fibroblasts cultured alone (□) or with CD34⁺ cells (▪) were collected at the indicated times and assayed for cytokines. The limit (dotted line) of cytokine detection and accuracy by ELISA is shown. Results are the mean ± SEM of four separate experiments.

Low and consistent surface expression of c-kitL and Flt3L was monitored on splenic fibroblasts by flow cytometry (Fig. 4⇓a). Unexpectedly, significant surface IL-15 expression was also observed. Similarly to c-kitL and Flt3L, high levels of IL-15 expression were detected after permeabilization of the cell membrane, indicating that splenic fibroblasts constitutively expressed IL-15 both on the cell surface and intracellularly. Splenic fibroblasts express IL-15R α-chain. To determine whether the expression of IL-15 on the cell surface was membrane-bound or secreted bound IL-15, cells were treated with neutralizing anti-IL-15 from the start of culture to day 3, when IL-15 was barely detectable in culture supernatants. No surface IL-15 expression was revealed in treated cells (data not shown), indicating that the IL-15 expressed on the surface of fibroblasts was secreted, rather than unsecreted, bound IL-15. After fixation, IL-15/IL-15Rα interaction appeared stable because treatment with anti-IL-15Rα mAb did not block bound IL-15 (Fig. 4⇓b).

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FIGURE 4.

Expression of c-kitL, Flt3L, and IL-15 in human spleen-derived fibroblasts. a, Nonpermeabilized (non perm.) and permeabilized (perm.) cells were stained with the indicated labeled mAbs (thick histograms) or with isotype-matched control Abs (thin histograms) after 4 days of culture. b, Cells were incubated with the anti-IL-15Rα mAb M162 (2 μg/ml) or with an isotype-matched control Ab (IgG1) for 24 h, washed, then stained with anti-IL-15 mAb (thick histograms) or control Ab (thin histograms).

Role of fibroblast-derived IL-15 in the production of NK cells

To evaluate the importance of the three cytokines, IL-15, Flt3L, and SCF, in the NK differentiation process stimulated by spleen-derived fibroblasts, neutralizing mAbs directed at each cytokine were added to the cultures for 3 wk, and cells with the NK phenotype were monitored on the basis of CD56 expression. Under conditions using a combination of the three neutralizing mAbs, no CD56⁺ cells could be detected (Fig. 5⇓a). However, when used individually, neutralizing anti-IL-15 mAb was found to block CD56⁺ cell development, whereas incubation with anti-SCF or anti-Flt3L mAbs did not.

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FIGURE 5.

Implication of IL-15 in NK cell production with splenic fibroblasts. a, Percentage of CD56⁺ cells cultured with neutralizing Abs to IL-15, Flt3L, and SCF for 3 wk. b, The number of nonadherent viable cells and percentage of CD56⁺ cells recovered from Transwells after 3 wk. Results are representative of two separate experiments.

To determine whether the IL-15 exposed on the surface of splenic fibroblasts and that produced extracellularly were both implicated in NK cell development, cocultures of fibroblasts were performed in Transwell plates without cell contacts with CD34⁺ cells. Similar numbers of nonadherent viable cells were monitored after 3 wk in cultures with and without cell-to-cell contacts (Fig. 5⇑b). Despite the lack of CD34⁺ cells (data not shown), no CD56⁺ cells were recovered from the inserts, whereas a fraction (17%) of highly expressing CD56⁺ cells was detected under cell-to-cell contact standard culture conditions. Moreover, when exogenous rIL-15 was added to CD34⁺ cells at 50 ng/ml, a concentration shown to elicit NK cell differentiation in stroma-free cultures (14), CD56⁺ cells were produced at levels comparable to those of cell-to-cell contact standard culture conditions (data not shown). All these results indicated that contact with fibroblasts and bound IL-15 interaction were necessary and sufficient for CD34⁺ progenitors to differentiate along the NK cell lineage.