Differential regulation of NK cell proliferation by type I and type II IFN
Matthew J. Loza1 and
Bice Perussia1
1 Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA
Correspondence to: B. Perussia; E-mail: Bice.Perussia{at}mail.tju.edu
Transmitting editor: R. Schreiber
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Abstract
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IFN, produced during viral infections by accessory (type I IFN) or NK cells (type II IFN), play a primary role in the regulation of immune and anti-viral NK cell effector functions. Because IFN have anti-proliferative effects on several cell types, including hematopoietic cells, we asked whether they modulate proliferation of human NK cells, and whether IFN-
and IFN-
mediate distinct effects on NK cells at different developmental stages. Analysis of proliferation at the single-cell level in human NK cells indicated that both IFN types inhibit IL-4-induced accumulation of immature CD56 IL-13+ NK cells in freshly separated peripheral blood lymphocytes and in cells derived from them after short-term cultures. However, IFN-
inhibited specifically the IL-4-dependent proliferation of these cells without affecting the IL-2-dependent one or that of the IL-13 cells, whereas IFN-
attenuated proliferation of NK cells at any developmental stage (both immature CD56IL-13+ and mature CD56+IL-13 IFN-
+ NK cells) and contributed to their monokine-induced differentiation to IFN-
-producing cells. Adding to our previous report that IL-13 inhibits accumulation of mature IFN-
+ NK cells, the present data unravel a mechanism by which peripheral immature IL-13+ and mature IFN-
+ NK cells can negatively regulate each others accumulation.
Keywords: cytokine, NK cell development
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Introduction
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Accessory cells first interacting with pathogens produce cytokines that activate NK cell cytotoxic effector functions and stimulate them to initiate inflammatory immune responses via IFN-
production. Among these, IL-12, produced after the initial wave of IFN-
and tumor necrosis factor (TNF)-
by monocyte/macrophages and dendritic cells (DC) [reviewed in (1,2)], plays a primary role as the most potent inducer of IFN-
production (3). IFN-
, primarily produced by plasmacytoid DC upon viral infection (4), enhances cytotoxicity and initiates efficient inflammatory type 1 responses by increasing expression of the signal transducing subunit of the IL-12 receptor (IL-12Rß2) (5), also regulated by IL-18 (6), and inducing production of IL-15 (7), a cytokine allowing proliferation/survival of both T and NK cells.
Peripheral NK cells in humans include a major CD56+ population and a minority of CD56 cells that produce IL-13, about one-third of which are also IL-5+ (8). IL-13+ NK cells are immature and do not produce IFN-
(8,9). In the presence of IL-12, they develop to lose ability to produce type 2 cytokines, while gaining that to produce IFN-
and phenotypic/functional characteristics of mature NK cells (9,10). Like relatively immature hematopoietic cells of other lineages, and unlike mature IFN-
+ NK cells, the IL-13+ NK cells have high proliferative potential. Preferential proliferation of a proportion of these IL-13+ cells accounts for the accumulation of type 2 cytokine+ NK cells in response to IL-4 (8,9), a cytokine capable of inhibiting the effects of IL-12 on IFN-
production and regulation of NK cell terminal differentiation (8). In the presence of IL-2, instead, part of the CD56IL-13+ cells partially mature to become CD56+, remaining IL-13+IFN-
. The population still CD56 in these cultures may contain cells that, phenotypically identical to most peripheral IL-13+ NK cells, are possibly less functionally mature.
Human NK and T cell terminal development, including that of the
-galactosyl ceramide-reactive NKT population (11), is strikingly similar and the sequence reported to occur in vitro for human cells has been confirmed to occur in vivo during NKT cell thymic development (12). Terminal T cell development is recapitulated in vitro using a combination of four accessory cell-produced monokines (IL-12, IL-18, IFN-
and IL-15). Each of these affects NK cell functions, e.g. IFN-
enhances cytotoxicity; IL-18 and IL-2 combined enhance IFN-
production (6); and, in the mouse, type I IFN are reportedly involved in inducing NK cell proliferation in vivo (13).
IFN-
, unlike IFN-
, has no consistent effect on NK cell functions (14), but like IFN-
it mediates anti-proliferative effects on numerous cell types, including immature hematopoietic cells of several lineages (15,16). An inhibitory effect of IFN-
on immature NK cell proliferation, and possibly facilitated differentiation to mature effector IFN-
+ NK cells, may, in part, explain the observation that NK cells from IFN-
/ mice produce levels of IL-13 higher than those produced by the IFN-
-sufficient parental strains (17). Here, we asked whether IFN modulate accumulation of human peripheral blood NK cells at different developmental stages by exerting anti-proliferative effects and/or contributing to their differentiation to IFN-
+ cells, testing the hypothesis that, like in T cells, IFN-
in combination with the monokines IL-12, IL-18 and IL-15 facilitates terminal NK cell differentiation.
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Methods
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Cell populations and culture conditions
Peripheral blood lymphocytes (PBL) were obtained from healthy adult and neonatal (umbilical cord) blood as described (18). Homogeneous polyclonal CD3CD161+CD56 immature NK cells populations were obtained from PBL by negative selection after a two-step purification protocol involving sequential depletion of: (i) most mature T, B, NK cells and monocytes using a mixture of mAb to leukocyte differentiation antigens (CD3, CD4, CD5, HLA-DR, CD56 and NKp46) and indirect anti-Ig rosetting (18), and (ii) possible residual mature NK cells by indirect immunofluorescence with anti-CD16 mAb and cell sorting with an Epics Elite flow cytometer (Beckman Coulter, Miami, FL) as previously described (9). These cells were used after a 2-week culture with IL-2, IL-12-neutralizing mAb, phytohemagglutinin (Sigma, St Louis, MO)-L, and 50-Gy irradiated Daudi B lymphoblastoid, autologous and allogeneic mononuclear cells [culture conditions detailed in (9)]. PBL and the CD56 immature NK cell populations were maintained in culture for 58 days in RPMI 1640 (Biowhittaker, Walkersville, MD) supplemented with L-glutamine, 5% autologous plasma, recombinant human cytokines and anti-cytokine mAb, as indicated. Purified human rIFN-
, 500 U/ml [IFN-A/D (BglII)], was from PBL Biomedical (New Brunswick, NJ); rIFN-
from Escherichia coli, 500 U/ml, was provided by Dr H. M. Shepard (Genentech, South San Francisco, CA); and the neutralizing anti-IFN-
mAb used were B133.1 and B133.5 (19). All other cytokines and mAb used were as previously reported (20).
Induction of cytokine production and intracellular cytokine detection
Cytokine production was induced in fresh and cultured lymphocytes upon a 5-h stimulation with Ca2+ ionophore A23187 (0.2 µg/ml), phorbol myristate acetate (PMA, 2 nM) (both from Sigma) and rIL-2 (100 U/ml) [Brefeldin A (Sigma) added, 10 µg/ml, during the last 3 h]. Stimulation, and combined detection of surface phenotype and intracellular IL-13, IL-5 and IFN-
accumulation were according to our previously reported protocols of multiple-color immunofluorescence (9). The total NK cell population was identified based on the combined expression of CD56 and CD161 [B159.5 + B199.2 mAb, both biotin-labeled, detected with streptavidinCyChrome (PharMingen, San Diego, CA)] and absence of CD3 [OKT3FITC or CD3PE/Texas Red (Caltag, Burlingame, CA)]. Viable lymphocytes were gated on the basis of forward and side angle light scatter characteristics, and flow cytometric analysis was performed on gated CD3(CD161/CD56)+ (total) or CD3(CD161/CD56)+IL-13+ NK cells, as indicated [specifics of controls and detection conditions used were as in (21)].
Normalization of the proportions and numbers of IL-13+ cells
When indicated, proportions and numbers of IL-13+ NK cells in lymphocytes from the various culture conditions were expressed relative to those, assigned an arbitrary value of 100% (0-fold change), in the corresponding control IL-2 cultures including control or IFN-
-neutralizing mAb, as indicated. Fold change of IL-13+ cells in experimental cultures = (%IL-13+ cellsexperimental culture conditions % IL-13+ cellscontrol cultures)/% IL-13+ cellscontrol cultures. Numbers of viable cells before and after culture were determined using Erythrosin B vital dye.
Carboxyfluorescein diacetate succinimidyl ester (CFSE) analysis
To determine the numbers of divisions that cytokine+ NK cells have undergone during culture, lymphocytes were cultured after labeling with CFSE (22) (0.250.75 µM, 107 cells/ml, 8 min, 37°C; Molecular Probes, Eugene, OR) as described (9). After culture, CFSE content, intracellular IL-13 accumulation and surface phenotype were simultaneously analyzed by immunofluorescence (flow cytometry) in gated CD3(CD56/CD161)+ cells according to previously published protocols (9). As previously described (9,23), based on viable cell counts and CFSE analysis, the minimum number of cells in the original population from which the IL-13+ NK cells detected at the end of the culture derived (minimum progenitor cell number) was calculated as (CFSEn+ cell number/2n) + (CFSEn + 1+ cell number/2n + 1) + ..., where n = division number, based on the discrete CFSE peaks. Details of the required (not shown) controls performed are given in (9). The calculation itself assumes no loss of the cells of interest, due to cell death or differentiation-induced changes in the original cell phenotype and/or function during culture (e.g. loss of IL-13 production). However, cell loss (of precursor cells and/or their potential progeny) can be determined and this is essential for correct interpretation of CFSE analysis when the calculated minimum progenitor cell number is lower than that of the corresponding cells in the original population. Number of cells lost is calculated as: n = (cell number in the original population progenitor cell number) x (average number of divisions the remaining cells have undergone).
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Results
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IFN-mediated inhibition of IL-4-induced accumulation of type 2 cytokine+ NK cells
As previously reported, most peripheral NK cells produced low levels of IFN-
, but distinct proportions of NK cells, corresponding to functionally immature CD56 and mature CD56+ cells (9), were detected producing respectively high levels of IL-13 (IL-13+) or IFN-
(IFN-
+/hi) upon stimulation of freshly separated PBL [(8) and not shown]. About one-third of the cells in the IL-13+ population also produced IL-5 [(9) and not shown]. Consistent with previous observations (8), the proportions of IL-13+ cells remained unchanged in NK cells derived from an 8-day culture of lymphocytes with IL-2 (Fig. 1A), and increased after culture with IL-2 and IL-4 compared to those from control cultures with IL-2 only. This increase was inhibited upon addition of IFN-
.

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Fig. 1. Effect of IFN- on IL-4-induced IL-13+ NK cell accumulation. PBL were cultured for 8 days with IL-12-neutralizing mAb, and the indicated cytokines and mAb (anti-IFN- = IFN- neutralizing mAb; control = isotype-matched TNF- non-neutralizing mAb). Cells were then stimulated and analyzed for IL-13 production within gated CD3(CD56/CD161)+ NK cells, as described in Methods. (A) IL-13 accumulation detected in three-color immunofluorescence in NK cells from stimulated lymphocytes after culture in the indicated conditions (representative experiment). (B) Top panel: proportions of IL-13+ NK cells in experiments performed with lymphocytes from eight (adult and neonatal) individual donors (mean ± SD of the results indicated at the right). *P < 0.001 between the indicated populations (paired Students t-test), regardless of lymphocyte origin (adult or neonatal blood). Bottom plots report proportions (%, left) and numbers (n, right) of IL-13+ NK cells in all experiments, normalized to those in control, IL-2 only, cultures (0-fold change), see Methods and (8,9). Identical symbols indicate cultures from the same individual. (C) Fold changes in proportions (left) and numbers (right) of IL-13+ NK cells after culture in the indicated conditions, relative to those in cultures with IL-2 + control mAb.
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Given the inter-individual variability of the IL-13+ cell proportions and numbers among freshly isolated lymphocytes and cells cultured with IL-2 + IL-4 (Fig. 1B, top), the data were analyzed after normalizing proportions (and numbers) of IL-13+ NK cells after culture in the experimental conditions to those in the respective control (IL-2 only) cultures, as described in Methods and previously detailed (8,9) (Fig. 1B, bottom and C). Addition of IFN-
to cultures with IL-2 only did not result in decreased proportions or numbers of IL-13+ NK cells (not shown). Instead, in the presence of IFN-
, the (IL-2 + IL-4)-induced increases in IL-13+ (and IL-5+, not shown) NK cell numbers were almost completely inhibited compared to those in the respective control cultures, indicating that IFN-
inhibits accumulation of type 2 cytokine+ NK cells. Accumulation of IL-13+ NK cells in cultures of freshly isolated lymphocytes with IL-4 depended in part on the IL-4-induced inhibition of IFN-
production by the CD56+ NK cells (8), since higher proportions and numbers of IL-13+ NK cells were detected, irrespective of the presence of IL-4, when endogenously produced IFN-
was neutralized (Fig. 1B and C) using anti-IFN-
mAb concentrations inhibiting the ability of exogenously added IFN-
to prevent IL-13+ NK cell accumulation (not shown). Inclusion of IL-12-neutralizing mAb in all cultures excluded participation of this monokine, possibly induced by IFN-
in residual monocytes and DC, to the observed effects. IFN-
also inhibited IL-13+ (and IL-5+, not shown) NK cell accumulation in cultures with IL-4 (Fig. 2). This was independent of IFN-
-induced endogenous IFN-
production, as IFN-
neutralization did not abrogate the inhibition. Addition of IFN-
to cultures with IL-2 only did not result in decreased proportions of IL-13+ NK cells, although their numbers were decreased by a factor similar to that for the total NK cells.

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Fig. 2. Effect of IFN- on IL-4-induced IL-13+ NK cell accumulation. Lymphocyte culture and cytokine detection were as in Fig. 1(A). Fold changes in proportions (left) and numbers (right) of IL-13+ NK cells after culture in the indicated conditions, relative to those in cultures with IL-2 + control mAb. Identical symbols indicate cultures from the same individuals.
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IFN-induced inhibition of NK cell proliferation
Both IL-2 and IL-4 induce NK cell proliferation, but IL-4 induces preferential proliferation of the IL-13+ NK cells within the total population (9). Similar percentages of cells in the total and the IL-13+ NK cells from cultures of freshly isolated lymphocytes with IL-2 only (Fig. 3) had undergone the same numbers of divisions, as determined by CFSE analysis at the single-cell level. As expected, in cultures with IL-4, the proportions of IL-13+ NK cells that had undergone the highest numbers of divisions were substantially increased, whereas those in the total NK cell population were decreased. In cultures with IL-12, which prevents IL-13+ NK cell accumulation (8) by inducing their differentiation (9), the percentages of both total and IL-13+ NK cells that had undergone multiple divisions were similarly decreased. The lack of IL-13+ NK cell accumulation in cultures with IL-12 (not shown) was mostly independent of induced IFN-
production, since IFN-
neutralization minimally affected it.

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Fig. 3. Effects of IL-4 and IL-12 on total and type 2 cytokine+ NK cell proliferation. CFSE-labeled neonatal lymphocytes were cultured (8 days) with IL-2, and the indicated cytokines and mAb (anti-IL-12 = IL-12-neutralizing mAb). Stimulation and analysis of intracellular IL-13 accumulation and CFSE content within NK cells is as described in Methods. Left: density plots of CFSE (x-axis) and IL-13 accumulation (y-axis). Figures at the top are division numbers. Division 0 was established based on fluorescence intensity in control CFSE-labeled cells cultured under the same conditions in the presence of mimosine to inhibit proliferation; division 6 corresponds to the fluorescence intensity in CFSE non-labeled cells cultured under the same conditions (9). Right: the histograms report percentages of cells that have undergone the indicated number of cell divisions (x-axis) in the total (top) and IL-13+ (bottom) NK cell populations (y-axis). Figures in parentheses are absolute numbers (x104) of the cell populations after culture in each condition. Experiments representative of three with similar results (two adult and one neonatal lymphocyte samples).
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To determine whether inhibition of proliferation accounts for the IFN-associated decrease in IL-13+ NK cell accumulation, the numbers of divisions undergone by NK cells were compared between lymphocytes from cultures with IL-2 + IL-4 without (control) and with added IFN-
or IFN-
(Fig. 4). Neutralization of endogenously produced IFN-
resulted in modestly and minimally increased numbers of divisions by the IL-13+ and total NK cells respectively. The numbers of divisions undergone by the IL-13+ cells, or those undergone by both these and the total NK cells, were decreased after culture with added IFN-
and IFN-
respectively.

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Fig. 4. IFN-mediated effects on IL-2- and IL-4-induced IL-13+ NK cell proliferation. Cells from PBL cultures with IL-2, IL-12-neutralizing mAb, and the indicated cytokines and mAb (anti-IFN- and control mAb as in Fig. 1) were analyzed as in Fig. 3. Data are percentages of cells in the total (open) and IL-13+ (filled symbols) NK cell populations (y-axis) that have undergone the indicated number of cell divisions (x-axis). Figures in parentheses are absolute numbers (x104) of the indicated NK cell populations after culture in each condition. The original numbers (x104) of total and IL-13+ NK cells were respectively 59 and 0.06. Experiment representative of five with similar results (two adult and three neonatal lymphocyte samples).
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After culture with IL-4 and added IFN-
-neutralizing mAb or IFN-
, the numbers of total NK cells was about one-third those in the original population. In both cultures, this corresponded to a loss of
75% of the cells from which the viable cells in culture could have derived, calculated as indicated in Methods. Such loss is possible from pre-existing NK cells and/or from NK cells newly accumulated following proliferation. Similar calculations indicated that, although the IL-13+ NK cell numbers had increased 5.7- and 3.6-fold in the cultures with added IFN-
-neutralizing mAb or IFN-
, 60 and 40% of these cells had been lost respectively in the two culture conditions. The greater loss (75%) of total NK cells versus IL-13+ NK cells in the presence of IFN-
(40%) suggests that IFN-
inhibits proliferation of the IL-13+ cells rather than induces their differentiation and/or death. Instead, after culture with IFN-
, 85% of both total and IL-13+ NK cells had been lost. Given the 50% decrease of IL-13+ NK cells from their original numbers, this corresponded to a loss of IL-13+ cells 40% greater than that of the corresponding population in culture without IFN-
, suggesting that IFN-
, in addition to inhibiting IL-13+ NK cell proliferation, prevents survival of both these and total NK cells to a similar extent or induces loss of ability to produce IL-13. Thus, the two IFN types prevent accumulation of the IL-13+ NK cells in response to IL-4 via distinct mechanisms, with only IFN-
specifically inhibiting proliferation of the immature IL-13+ cells.
IFN-induced inhibition of functionally immature type 2 cytokine+ NK cells
IL-13+ NK cells include an immature CD56IFN-
and an intermediate CD56+ population, not necessarily IFN-
+ (9). To determine whether IFN affect proliferation of NK cells at specific developmental stages, experiments similar to the above were performed with cells derived from cultures of immature homogeneous CD161+CD56 IL-13+ NK cell populations with IL-2 (Fig. 5). After culture, the cells, still CD161+, contained CD56 and CD56+ cells, both including IL-13+ cells (20 and
1% respectively; not shown). Upon further culture with IL-2 + IFN-
-neutralizing mAb and IL-4, the percentages of cells that had undergone several rounds of divisions were increased, compared to those in cultures with IL-2 only, in the IL-13+ and, to a lesser extent, in the IL-13 immature cell population still CD56. Proliferation of the total and the IL-13+ cells was not inhibited by exogenously added IFN-
, whereas that of both populations was decreased in cultures with added IFN-
, resulting in lower proportions of cells that had undergone high division numbers. No, or minimal proportions of, cells had undergone more division rounds in the total CD56+ and the CD56+IL-13+ cells in the presence of IL-4. Added IFN-
, but not IFN-
, resulted in decreased proportions of CD56+IL-13+ cells in the highest division peaks, similar to that observed in the cells still CD56. The results were similar in cultures without added IL-4, confirming that, unlike IFN-
, IFN-
does not attenuate proliferation of more mature NK cells. [3H]Thymidine incorporation assays in similar experiments gave analogous results (not shown). In all experiments described up to here the same results were obtained in cultures where IL-15 was used instead of IL-2 (not shown).

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Fig. 5. IFN-mediated effects on immature NK cell proliferation. Immature NK cells (obtained from CD3CD61+CD56 neonatal lymphocytes, see Methods) were labeled with CFSE and cultured for 5 additional days with IL-2, and the cytokines and mAb indicated at the right. After culture, the cells were stimulated, and IL-13 production and CFSE content were analyzed within gated CD3CD61+CD56 or CD3CD56+ lymphocytes as described in Fig. 3. Data presentation is as in Fig. 3. Experiment representative of three with similar results.
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Effect of monokines on immature NK cell differentiation
Immature CD56IL-13+ NK cells are induced to differentiate to CD56+IFN-
+ cells in culture with IL-12 and B lymphoblastoid cell lines (9). Immature, CD2/loIL-13+ T cells are similarly induced to differentiate in cultures with IL-12 and TCR-mediated stimuli, concomitant with induced CD56 expression (8,20). The monokines IL-12, IL-15 and IL-18 combined with IFN-
suffice to induce the same effect substituting for TCR-mediated stimulation, and, as expected, T cell differentiation is accompanied by decreased proliferation (20). T and NK cells undergo the same developmental sequence, and IFN-
attenuates proliferation of NK cells at any developmental stage. Consequently, we asked whether, like for TCR stimulation, IFN-
in combination with IL-12 and IL-18 could substitute for feeder B cells to induce differentiation of homogeneous CD56 immature NK cell populations (Fig. 6). IL-15 was included in all cultures to maintain NK cell survival. The NK cell numbers, compared to those in the original cell populations, increased
3-fold in all cultures without IL-12, were
5075% higher in those with IL-12 (including those with added IFN-
or IL-18) and decreased by
20% in those with all monokines combined (not shown). The numbers of IFN-
+ cells in cultures with added IFN-
or IL-18 individually were similar to those in control cultures with IL-15 only, but increased after culture with IFN-
+ IL-18, mostly depending on increased numbers of IL-13+IFN-
+ cells. The numbers of IFN-
+ cells were higher in all cultures with IL-12 added, despite reduced total NK cell numbers. The remaining IL-13+ cells in these cultures were mostly IFN-
+, with a few still IFN-
. The increased numbers of IL-13+ cells in the cultures with IFN-
, but no IL-12, did not correspond to proportions of IFN-
+ cells significantly greater than those in the original population. The presence of IFN-
, per se, did not result in specific loss of immature IL-13+ NK cells. With IL-12 added, the IL-13+ NK cell numbers decreased in all culture conditions, with the highest proportions of IL-13+IFN-
+ cells detected when IL-18 and IFN-
were combined. IL-18, which had no effect on the proportions, numbers and accumulation of IL-13+ NK cells in cultures of freshly separated lymphocytes with or without IL-2 or IL-2 + IL-4, also had no effect on the proliferation of immature and mature NK cells (not shown). As expected in the case of induced differentiation, a proportion of the cells expressed CD56 after culture with the monokine combination (not shown). Thus, the same set of monokines can induce both T and NK cell differentiation from IL-13+ to IFN-
+ cells; IFN-
, inefficient alone, cooperates with IL-12 and IL-18 to maximize this process, still maintaining the highest numbers of type 2 cytokine+IFN-
+ NK cells.
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Discussion
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Our data indicate that both IFN-
and IFN-
can inhibit IL-2 + IL-4-induced accumulation of type 2 cytokine+ NK cells, but, importantly, they diverge in the NK cell specificity of their effect. IFN-
attenuates proliferation of all (IL-13 and IL-13+) NK cells both in freshly isolated lymphocytes and in cells derived from immature CD56 NK cell cultures, and facilitates NK cell differentiation to effector IFN-
+ cells; IFN-
specifically inhibits proliferation of the immature CD56 IL-13+ NK cells without affecting the predominant CD56+ IL-13IFN-
+/lo population in fresh peripheral blood. The overall effects of IFN on accumulation of IL-13+ NK cells are consistent with the prediction from the NK cell developmental pathway from proliferative immature type 2 cytokine+ to non-proliferative mature type 1 cytokine+ cells (8,9) that: factors (IFN-
) solely inhibiting proliferation of immature cells lessen IL-13+ cell accumulation without affecting development or modifying the proportions of IFN-
+ cells; factors (IFN-
) increasing NK cell responsiveness to IL-12 induce IL-13+ NK cell loss, and concomitantly allow their differentiation (substituting, in conjunction with the monokine combination IL-12 + IL-15 + IL-18, for B feeder cells) and induce increased proportions of IFN-
+ (IL-13+ or IL-13) cells.
Our observation that IFN-
inhibits proliferation of human NK cells at any developmental stage is in agreement with in vitro data in murine NK cells, and may seem to contrast with the data in the same report indicating a role of IFN
/ß in the in vivo proliferation of NK cells in mice treated with polyinosinicpolycytidylic acid or infected with murine cytomegalovirus (13). However, it has been reported that type I IFN-induced IL-15 is responsible for the induced in vivo proliferation (24) and the available experimental evidence does not allow us to exclude the possibility that the proliferation level of NK cells in vivo would have been higher in the absence of sustained IFN-
/ß levels. The data indicating that human NK cell proliferation in response to IL-15 or IL-2 in vitro is maintained, although significantly attenuated, in the presence of type I IFN, support the testable hypothesis that IFN-
/ß-dependent NK cell proliferation may occur in vivo only in selected situations. These would correspond to those in which relatively low levels of IFN-
/ß are produced by the accessory cells involved, relatively high IL-15 levels are produced to fully sustain NK cell proliferation after initial production of transiently high levels of IFN-
/ß and the response of T cells (possibly competing with NK cells for IL-15 for proliferation) is not overwhelming.
The observation that neutralization of IFN-
produced in response to IL-2 or IL-15 restores proliferation of IL-13+ NK cells indicates that physiologically relevant levels of IFN-
are effective to inhibit peripheral type 2 cytokine+ NK cell accumulation. Although IFN-
inhibited specifically proliferation of the IL-13+ NK cells, its effects were minimal on those IL-13+ cells still CD56 after culture of peripheral immature CD56 NK cells. These apparently contrasting results are reconciled considering that the latter cells are likely functionally less mature than most peripheral blood CD56 IL-13+ cells. This is supported by the observations that, unlike other NK cell populations, these cells constitutively produce detectable IL-13 levels (8) (possibly in response to undefined physiologic stimuli), and proliferate in response to IL-2 and/or IL-4 more than the IL-13+CD56+ cells differentiated in the same cultures and the freshly separated CD56 ones. Alternatively, differences in the response of freshly isolated and cultured immature NK cells to IFN-
might depend on lack of IFN-
-responsive accessory cells in the latter population. An IFN-
-inducible factor produced by accessory cells [e.g. the type I IFN-
(25)] may mediate the block in proliferation. However, IFN-
affects only the IL-13+ population, making this possibility unlikely, unless the IL-13+ cells are more sensitive than the IL-13 ones to low levels of the putative IFN-
-induced factor(s). Whichever the mechanism, the observation that IFN-
inhibits the IL-4-induced, but not the basal or IL-2-induced, proliferation of NK cells is consistent with its lack of effect on most CD56+ NK cells, in which both the IL-13 and the minor IL-13+ NK cell population have minimal proliferative response to IL-4.
The molecular bases for the differential effects of IFN-
and IFN-
on IL-13 NK cells are to be defined. IL-4 receptor engagement activates several signaling pathways, including JAK1,3/STAT6, IRS1 and 2, Grb, and Shc (26). Differences in developmental stage-dependent expression of IL-4 receptor-associated signaling molecules may dictate the extent of proliferative response to IL-4 in immature IL-13+ and mature IL-13 NK cells. Whether IL-4 signaling in the mature CD56+ cells involves pathways distinct from those affected by IFN-
in the IL-13+ NK cells remains to be determined.
The differential ability of the two IFN types to inhibit NK cell proliferation is a likely basis for complementing functions of IFN-
and IFN-
in inflammatory responses. IFN-
is one of the first inflammatory mediators released by accessory cells in response to pathogens and/or tissue damage (14). In an inflammatory reaction involving IFN-
production (e.g. viral infection), IFN-
, by inhibiting proliferation of most peripheral immature/intermediate IL-13+ cells (CD56 and CD56+) and sensitizing them to the effects of IL-12 via induced expression of significant levels of functional IL-12R (5), would function to prime them to proceed to terminal differentiation in response to other maturation stimuli (i.e. IL-12 and IL-18 for both NK and T cells), concomitantly enhancing the effector functions of the terminally differentiated cells [e.g. cytotoxic potential of NK cells; antigen presentation by accessory cells; IFN-
and IL-15 production by monocytes and IFN-
by NK and T cells; reviewed in (14)]. IFN-
is produced and acts at a later phase. By specifically inhibiting the default accumulation of residual immature lymphocytes that, via type 2 cytokine production, contribute to maintain monocytes in a relatively immature state, it would function to increase the efficacy of cell-mediated immune responses, concomitantly inducing maturation of accessory cells.
IL-13 plays a primary role in allergic asthma-associated pathology [(27), reviewed in (28)] and IL-13+ (IL-5+) NK cells are present in the mononuclear cell population found in the bronchoalveolar lavage from allergic asthmatic individuals analyzed ex vivo immediately after in vivo challenge with the relevant allergen (29). Also, NK cell-derived IL-5 is critical in the pathogenesis of experimental allergic asthma in murine models (30) (IL-13 not tested). In the allergy context, NK cell-derived IL-13 may prevent progression of IFN-
-associated, type 1 immune responses, and thus contribute to allergic pathology by inhibiting accumulation and maintenance of the IFN-
+ NK cells independently of its ability to inhibit IL-12 production by accessory cells (8). As a result of inhibition of their differentiation to IFN-
+ cells, the contribution of immature NK cell-derived cytokines to atopic reactions may involve, in addition to IL-13, TNF-
, granulocyte macrophage colony stimulating factor and IL-5 (all expressed at the highest levels in immature NK cells) (10). These cytokines affect B cell survival (31) and Ig switching to non-inflammatory (IgG2) and Fc
R-triggering (IgE) isotypes/classes, the latter directly inducing mast cell degranulation, and promote mobilization and survival of myeloid cells (basophils, mast cells and eosinophils) pathogenic in allergic reactions (32).
We previously reported that IL-13 prevents accumulation of IFN-
+, but not total, NK cells (8). Here we show that IFN-
specifically prevents accumulation of the IL-13+ NK cells. Most peripheral NK cells produce either IL-13 or IFN-
exclusive of each other (8). Thus, a negative feedback loop can be established between immature and mature NK cells, and maintenance of peripheral NK cells at either developmental stage does not depend on autocrine functions of the respective NK cells, but is controlled by cytokines produced exclusively by their progenitors or their differentiated progeny. This is unlike T cells which, at any developmental stage, can maintain their own survival via production of autocrine growth factors, i.e. IL-2 and IL-4. Because T and NK cells share the same developmental pathway (11,20), IFN may similarly inhibit type 2 T cell accumulation. However, differences in responses to IFN are possible between type 2 CD4+ and CD8+ cells, since IL-4 enhances proliferation only of the latter (23), and between T and NK cells, depending on modulated expression and responsiveness of IFN receptors possibly induced in T cells upon TCR engagement.
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Acknowledgements
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This work was supported, in part, by USPHS grants AI55842, CA77401 (B. P.) and T32-CA09683 (M. J. L.). We thank Dr V. Berghella and the Staff of the Obstetrics and Gynecology Division, Thomas Jefferson Hospital, for providing the umbilical cord blood samples, and the personnel in the KCC Flow Cytometry Facility for assistance.
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Abbreviations
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CFSEcarboxyfluorescein diacetate succinimidyl ester
DCdendritic cell
PBLperipheral blood lymphocyte
PEphycoerythrin
PMAphorbol myristate acetate
TNFtumor necrosis factor
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