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IMMUNOBIOLOGY
From the Department of Internal Medicine, Division of
Hematology/Oncology; the Department of Molecular Virology, Immunology
and Medical Genetics, Division of Human Cancer Genetics; the Department
of Surgery; and the Comprehensive Cancer Center, The Ohio State
University, Columbus, OH; and BASF Bioresearch Corporation, Worcester,
MA.
During the innate immune response to infection,
monocyte-derived cytokines (monokines), stimulate natural killer (NK)
cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by
CD56 surface density expression (ie, CD56bright and
CD56dim). In this report, it is shown that
CD56bright NK cells produce significantly greater levels of
interferon- Natural killer (NK) cells are innate immune
effectors that produce immunoregulatory cytokines, such as interferon
(IFN)- CD56bright NK cells constitutively express the high-
and intermediate-affinity IL-2 receptors and expand in vitro and in
vivo in response to low (picomolar) doses of IL-2.6-8
These NK cells also express the c-kit receptor tyrosine
kinase whose ligand enhances IL-2-induced
proliferation.9,10 In contrast, resting
CD56dim NK cells express only the intermediate
affinity IL-2 receptor, are c-kitneg, and
proliferate weakly in response to high doses of IL-2 (1 to 10 nM)
in vitro, even after induction of the high-affinity IL-2
receptor.6,7 Resting CD56dim NK cells are more
cytotoxic against NK-sensitive targets than CD56bright NK
cells.11 However, after activation with IL-2 or IL-12,
CD56bright cells exhibit similar or enhanced cytotoxicity
against NK targets compared to CD56dim
cells.11-13
NK cell subsets have differential natural killer receptor (NKR)
repertoires. All resting CD56bright NK cells have high
expression of CD94/NKG2 C-type lectin receptors.14 A small
percentage (less than 10%) expresses killer cell immunoglobulin-like receptors (KIR),15 while most (more than 85%) resting
CD56dim NK cells are KIR+ and have low
expression of CD94/NKG2.
CD56bright NK cells also express the adhesion
molecule L-selectin (CD62L), which mediates initial interactions with
vascular endothelium.16 CD56dim NK cells lack
this receptor but have recently been found to express PEN5, an NK
cell-restricted sulfated lactosamine epitope that partially mediates
the binding of L-selectin,15 thus suggesting the
potential for differential trafficking of human NK cell subsets in
vivo. Therefore, CD56bright and CD56dim NK
cells differ in their proliferative response to IL-2, intrinsic cytotoxic capacity, NKR repertoire, and adhesion molecule expression.
NK cells constitutively express receptors for monocyte-derived
cytokines (monokines) and produce critical cytokines, such as IFN- Cell culture reagents and antibodies
Purification of human NK cell subsets and
macrophages
NK cell monokine and
PMA-ionomycin stimulation
Quantitation of cytokine transcripts by real-time RT-PCR FACS-purified CD56bright and CD56dim NK cells (1 × 105) were either immediately lysed for RNA (Qiagen RNeasy lysis buffer; Qiagen, Valencia, CA) or cultured at 1 × 105 cells/well with recombinant monokines. Cells were harvested at 24 hours and lysed with 300 µL RNA lysis buffer. Total cellular RNA was isolated (Qiagen RNeasy Mini-kits; Qiagen) and cDNA was generated with random hexamer primers and MMLV-RT according to the manufacturer's recommendations (Gibco Life Technologies, Rockville, MD). cDNA was then used as a template for real-time polymerase chain reaction (PCR).Real-time quantitative reverse transcription (RT)-PCR is a novel method
to accurately measure amplified target copy number through the use of a
dual-labeled fluorogenic probe.21 Real-time PCR reactions
for human IFN- NK cell and macrophage co-cultures Purified CD56bright and CD56dim NK cells (1.0 × 105) were co-cultured with autologous macrophages (1.0 × 105) as previously described22 and stimulated with 10 µg/mL lipopolysaccharide (LPS; serotype 0127 B8; Sigma, St Louis, MO) for 72 hours.Statistical analysis Statistical analysis was performed using the Student paired t test; P < .05 was considered significant.
CD56bright NK cells produce abundant type 1 and type 2 cytokines compared to CD56dim NK cells We stimulated sorted resting CD56bright and CD56dim NK cells (Figure 1) with the recombinant monokines IL-12, IL-15, IL-18, and IL-1 alone and in combination with IL-12 or
IL-15. To examine the stimulation of NK cells independent of monokine
receptor expression, NK cell subsets were also activated with phorbol
esters (PMA) plus ionomycin. The CD56bright subset produced
significantly more of the type 1 cytokines IFN- and TNF- than
CD56dim NK cells cultured under identical conditions after
stimulation with monokines or PMA plus ionomycin (Figure
2). CD56bright NK cells
co-stimulated with IL-18 plus IL-12 produced the most IFN-
protein (Figure 2A), whereas stimulation with IL-18 plus IL-15 or
IL-1 plus IL-15 induced the highest levels of TNF- protein production.
NK cell production of IL-10, a type 2 cytokine, was only detected after
co-stimulation with IL-12 plus IL-15 (Figure
3A), with CD56bright NK cells
producing in excess of 25-fold more IL-10 protein than CD56dim cells. Interestingly, other monokine combinations,
including IL-12 plus IL-18, failed to elicit any production of IL-10
from either subset. Modest amounts of IL-13, another cytokine produced by committed Th2 cells, were detected in cultures of
CD56bright NK cells stimulated with IL-15 plus IL-18 or
IL-1
Thus, CD56bright NK cells produce high levels of 2 principal type 1 cytokines, IFN- CD56bright NK cell
production of other pro-inflammatory cytokines, GM-CSF,
and TNF- . Unlike
other NK-derived cytokines, stimulation with PMA plus ionomycin induced
the highest levels of GM-CSF protein from CD56bright NK
cells. CD56bright NK cell production of the
pro-inflammatory cytokine TNF- , after culture with monokines or PMA
plus ionomycin, was modest (less than 300 pg/mL) and somewhat variable,
but it was consistently greater than in the CD56dim NK cell
subset (data not shown).
Cytokine transcript levels in NK cell subsets To determine whether NK cell subsets have differential baseline expression of cytokines not detectable by ELISA that might account for observed differences in protein production, we measured transcript levels of 3 primary NK-derived cytokines in resting and monokine-activated NK cell subsets by real-time quantitative RT-PCR. Resting subsets lacked any detectable expression of IL-10 (data not shown), but both CD56bright and CD56dim NK cell subsets expressed equal amounts of IFN- and GM-CSF transcript (data
not shown; n = 3). Therefore, there is no detectable difference in
the baseline production of these cytokine transcripts by resting NK
cell subsets. After 24 hours of monokine stimulation, the
CD56bright NK subset produced higher levels of IFN- and
GM-CSF transcript than the CD56dim NK subset, consistent
with their production of the respective proteins (Figure
5A,B).
CD56bright NK cells
produce significantly more IFN- production by
human NK cells in vitro.22 To determine the subset of NK
cells responsible for IFN- production after monokine
activation by LPS, purified CD56bright and
CD56dim NK cells were co-cultured with LPS-stimulated
autologous macrophages (Figure 6).
Similar to results obtained with recombinant monokines, CD56bright NK cells produced 8-fold more IFN- protein
than CD56dim cells (n = 7). Thus, CD56bright
NK cells are the primary producers of IFN- in response to both recombinant and endogenous monokines.
Collectively, our results reveal that CD56bright human
NK cells are the primary source of NK-derived immunoregulatory
cytokines, including IFN- Peritt et al25 recently reported the differentiation of
human NK cells into NK1 and NK2 subsets by generating NK cell clones after 8-day in vitro culture under type 1- or type 2-inducing conditions. After stimulation with PMA plus ionomycin, NK1 cells produced IFN- Additional data presented here indicate that the qualitative and
quantitative production of monokines after host infection are likely
important in distinguishing the induction of type 1 and type 2 cytokines by CD56bright NK cells. IL-15 co-stimulation was
requisite for CD56bright NK cell production of type 2 cytokines (eg, IL-10 and IL-13), whereas IL-12 co-stimulation was
required for optimal production of the type 1 cytokine IFN- It has been hypothesized that CD56bright and
CD56dim NK cells represent different stages of NK cell
maturation, with CD56dim NK cells being the more
differentiated cell type.11 This paradigm is based on
observations that resting CD56dim NK cells are more
cytotoxic than CD56bright NK cells, express high surface
density expression of both KIR15 and CD16 (which mediate
natural NK cytotoxicity28 and antibody-dependent cellular
cytotoxicity,29 respectively), and do not readily
proliferate in response to IL-2. In the current study we provide new
evidence to suggest that CD56bright NK cells are the major
cytokine-producing subset of human NK cells, an attribute that is
associated with essential host function. Based on these data, we
propose that CD56bright and CD56dim NK cells
represent functionally and phenotypically distinct subsets of NK cells
with unique immunoregulatory roles in vivo (Figure 7).
Numerous studies from our laboratory and those of others have definitively identified IL-15 as the critical factor for the development of human and murine NK cells.30-32 Culture of CD34+Linneg human hematopoietic cells with IL-15 results in the differentiation of mature, functional CD56+ NK cells. Culturing progenitor cells with flt3 ligand (FL) or c-kit ligand (KL) can increase the NK cell precursor frequency through up-regulation of the IL-15R complex.30,33 However, in vitro-generated NK cells are consistently CD56brightCD16dim/negKIRlow/neg, making them more phenotypically similar to the minor (approximately 10%) population of CD56bright peripheral blood NK cells.33 Further, these cell also produce immunoregulatory cytokines. Until recently, evidence for the differentiation of distinct CD56bright and CD56dim NK cell populations has been lacking because there were no reports of in vitro-generated CD56dim NK cells, lending support to the theory that CD56bright NK cells may be the more immature cell type. The discovery of a novel cytokine, IL-21, by Foster et al34 may shed some light on the developmental differences between CD56bright and CD56dim human NK cell subsets. IL-21 is a unique cytokine most closely related to IL-2 and IL-15, with effects on B- and T-cell proliferation and NK cell differentiation, proliferation, and cytotoxicity. The authors cultured CD34+Linneg hematopoietic progenitors with FL ± IL-21 and FL + IL-15 ± IL-21. IL-21, in combination with IL-15 and FL, induced the differentiation of CD56+CD16+ NK cells, which, by flow cytometric analysis, appear to be CD56dimCD16+. FL plus IL-21 alone did not induce NK cell differentiation, whereas stimulation with FL plus IL-15 resulted in the expected population of CD56brightCD16neg NK cells.34 The discovery of this cytokine, which can, in combination with IL-15, induce differentiation of CD56dimCD16+ NK cells, supports a hypothesis whereby human CD56bright and CD56dim NK cells are terminally differentiated cell types that develop within the bone marrow under the influence of differential growth factors, such as IL-15 and IL-21. Further studies are required to investigate definitively the developmental relation between CD56bright and CD56dim NK cells. It will be interesting to determine the role of IL-21/IL-21R in the development of human NK cell subsets and NK receptors (eg, CD16 and KIR). As we continue to develop immunotherapeutic strategies that target human NK cells, such as the selective expansion of CD56bright NK cells with low-dose IL-2 treatment of malignancies8,35 and human immunodeficiency virus,36 it will be important to understand the functional differences between these human NK cell subsets. Knowledge of the distinct functional attributes of CD56bright (eg, immunoregulatory cytokine production) and CD56dim (eg, cytotoxicity and antibody-dependent cellular cytotoxicity) human NK cell subsets and the factors involved in their development and expansion may enable us to design strategies that preferentially activate that subset with the greatest therapeutic potential for a particular disease.
We thank A. Oberyszyn for cell sorting and J. Bush and A. Ponnappan for technical assistance.
Submitted November 3, 2000; accepted January 9, 2001.
Supported by National Institutes of Health grants CA-68458, CA-65670, and P30CA-16058. M.A. Cooper is a Howard Hughes Medical Institute Medical Student Research Training Fellow. T.A.F. is the recipient of the Medical Scientist Training Program and the Bennett Fellowships from The Ohio State University College of Medicine and Public Health.
M.A. Cooper and T.A.F. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael A. Caligiuri, Department of Internal Medicine, Division of Hematology/Oncology, The Ohio State University, 458A Starling-Loving Hall, 320 West 10th Ave, Columbus, OH 43210; e-mail: caligiuri-1{at}medctr.osu.edu.
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© 2001 by The American Society of Hematology.
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