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IMMUNOBIOLOGY
From the Department of Medicine, University of
Minnesota Cancer Center, Minneapolis, MN.
The stage of progenitor maturation and factors that determine the
fate and clonal acquisition of human natural killer (NK) cell receptors
during development are unknown. To study human NK cell receptor
ontogeny, umbilical cord blood
CD34+/Lin In contrast to T cells, natural killer (NK) cells
kill a broad array of targets in an HLA-unrestricted manner. However,
current observations counter the notion that NK cell killing is totally major histocompatibility complex (MHC) unrestricted.1-3
Although antigen presented by MHC class I molecules activates T cells, "self" class I presentation renders some targets resistant
to lysis by NK cells KIRs are encoded on chromosome 19 and designated by their number
of immunoglobulin domains (KIR2D, KIR3D, etc). Their inhibitory and
activating function is determined by the length of their associated cytoplasmic tail, long (L) or short (S),
respectively.11-13 At least 3 groups of inhibitory
receptors (KIR2DL1/S1, KIR2DL2/L3/S2, and KIR3DL1) have class I ligands
identified (cw4, cw3, and bw4).14-16 There has been much
progress in understanding the determinants of how class I and
associated binding peptides may influence receptor ligation17,18 and subsequent signaling.19-22
The lectin-type human NK cell receptors on chromosome 12 initially
described by Houchins et al23 and Yabe et
al24 were difficult to characterize because they exist as
heterodimers, disulfide-linked to CD94.25,26 It is now
well characterized that NKG2/CD94 family members are inhibitory (NKG2A,
NKG2B) and activating (NKG2C, NKG2E) but recognize nonclassical class
1b HLA-E molecules bound with peptide leader sequences from several classical ABC alleles as well as HLA-G.27-29 The crystal
structure of the extracellular domain of CD94 shows a unique c-type
lectin fold and a putative ligand binding region for HLA-E with the
NKG2/CD94 heterodimer.30 Recently, a unique role of NKG2D,
which does not bind CD94, has been elucidated.31,32 NKG2D
serves as an activating receptor after interaction with the
stress-inducible nonclassical MICA (and possibly MICB) class I homolog.
The ontogeny of CD94 lectin and immunoglobulin receptors during human
NK cell development and factors that determine NK cell receptor
repertoires are unknown.
NK cells are derived from human marrow primitive
progenitors.33,34 In the presence of interleukin-2 (IL-2)
alone, NK cell differentiation from these progenitors is dependent on
direct contact with human stromal ligands.35 The
development of NK cells from marrow progenitors has been corroborated
in vitro36-38 and in vivo after transplanting fetal sheep
with CD34+/Lin In mice, the ability of stroma to induce differentiation is, at least
in part, regulated by the transcription factor interferon-regulatory factor-1 transcription of IL-15.44 In human studies, IL-15
made by stroma and macrophages plays a role in NK development and
survival by interaction with components of the IL-2
receptor.38,45 NK cell differentiation from progenitors
requires primitive-acting factors like FL, KL, and
IL-3,34,40 and the need for lymphoid factors likely occurs
later and may coincide with IL-2/IL-15 receptor Despite progress in defining the role of cytokines in NK cell ontogeny,
differentiation of single human progenitors into NK cells was not
possible without murine stromal feeders.46 We have
extensively studied a murine cell line (derived from fetal liver)
called AFT024 initially described by Moore et al.47 AFT024 and defined cytokines differentiate adult marrow
CD34+/Lin Umbilical cord blood, normal bone marrow, and fetal liver
Purification of primitive progenitors
Culture of hematopoietic progenitors CD34+/Lin /CD38 cells
were plated in 24-well plates (Costar, Cambridge, MA) with a 2:1
(vol/vol) mix of Dulbecco modified Eagle medium (DMEM) high glucose/Ham
F12-based medium without stroma (Gibco Laboratories, Grand Island, NY),
in direct contact with the AFT024 stromal cell line, or in a Transwell
insert separating progenitors from stroma by a 0.4-µm collagen
membrane (Costar) as indicated. The DMEM/F12-based medium was developed
to maximize NK cell growth50 and is supplemented with 24 µM 2-mercaptoethanol, 50 µM ethanolamine, 20 mg/L ascorbic acid, 50 µg/L sodium selenite (Na2SeO3), 100 U/mL
penicillin, and 100 U/mL streptomycin (Gibco). Twenty percent
heat-inactivated human AB serum (North American Biologicals, Miami, FL)
was used at culture initiation that was reduced to 10% for subsequent
media changes. Cytokines were supplemented as indicated with 1000 U/mL
IL-2 (a gift from Amgen, Thousand Oaks, CA), 10 ng/mL IL-15 (R&D
Systems, Minneapolis, MN), 10 ng/mL FL (a gift from Immunex, Seattle,
WA), 20 ng/mL KL (or Stem Cell Factor, a gift from Amgen), and 20 ng/mL
IL-7 (R&D Systems). All cytokines were added fresh with weekly media
changes except for IL-3 (R&D Systems), which was added at 5 ng/mL only
once at culture initiation. Cultures were maintained in a humidified
atmosphere at 37°C and 5% CO2.
Antibodies and determination of absolute cell counts FITC, PE, peridinin chlorophyll protein, and APC coupled control immunoglobulins or specific antibodies directed at APC CD56 (BD Pharmingen, San Diego, CA), CD94 (FITC-conjugated, clone HP-3D9; BD Pharmingen; or PE-conjugated, clone HP-3B1; Beckman Coulter, Miami, FL), NKG2A (clone Z199; Beckman Coulter), CD158a (PE-conjugated, clone EB6; Beckman Coulter; or FITC-conjugated, clone HP-3E4, BD Pharmingen), CD158b (clone GL183; Beckman Coulter), and NKB1 (clone DX9; BD Bioscience) were used to evaluate progeny NK cells from differentiation cultures. Progeny of single cells and bulk cultures were harvested and divided into 3 to 4 aliquots to facilitate evaluation of 9 to 12 surface markers. Absolute cell numbers were determined by addition of 3 × 104 polystyrene microspheres (Polysciences, Warrington, PA) to each aliquot of cultured progeny. After gating out debris, absolute cell numbers were calculated, using the method described by Pribyl et al51 and by Larson and LeBien.52 The absolute number of cells per well was calculated as [(total number of beads added/well)/(number of beads collected) × (number of cells in the phenotype gate of interest) × (the number of times the initial sample was divided)]. All analyses were performed with a FACSCalibur and CELLQuest software (Becton Dickinson).Class I HLA typing of UCB cells In some experiments, the CD34 fraction was
collected from the CD34+ enrichment step and cryopreserved
for HLA typing. HLA typing was performed by a standard
complement-dependent cytotoxicity method by using local and commercial
serologic typing trays.
Statistics Results of experimental points obtained from multiple experiments were reported as mean ± 1 SEM. Significance levels were determined by 2-sided Student t test analysis.
UCB CD34+/Lin /CD38) along the NK
cell lineage.
Primitive-acting cytokines (FL, KL, and IL-3) and
lymphocyte-stimulating cytokines (IL-7 and IL-2) were required for NK
cell differentiation of adult BM progenitors. Because the cytokine responsiveness of UCB cells may be different from that of adult BM,53 in a first set of experiments, UCB
CD34+/Lin IL-15 or IL-2 and direct contact with AFT024 ligands are optimal for lectin receptor and KIR acquisition To determine the importance of factors produced by the AFT024 feeder, 300 UCB CD34+/Lin/CD38
cells were cultured in direct contact with AFT024 (Contact), separated
from AFT024 by a microporous Transwell (TW) membrane, or in the absence
of AFT024 (No AFT024). All cultures contained FL, KL, and IL-3 with or
without the addition of other cytokines (Figure
1). In the absence of IL-7, IL-15, or
IL-2, the absolute number of NK cells derived from 300 UCB progenitors
was less than 6000 and not different for any of the AFT024 conditions
(Contact, TW, or No AFT024). Addition of IL-7 to FL, KL, and IL-3
resulted in a small increase in NK cell differentiation. In contrast,
addition of IL-15 or IL-2 to FL, KL, and IL-3 resulted in a marked
increase in NK cell differentiation, which for each cytokine was
greatest when UCB progenitors were cultured in direct contact with
AFT024 ligands (Figure 1A).
Each of the above culture conditions was then analyzed for the absolute
number of NK cells expressing either CD94 (antibody clone HP-3B1) or a
KIR (using a PE-combined cocktail of antibodies, DX9, GL183, and EB6).
Both CD94- and KIR-expressing NK cells (Table 1 for KIR antibody and specificity) were
low and independent of AFT024 conditions when 300 UCB
CD34+/Lin
IL-15, IL-2, or IL-7 is needed for NK cell long-term proliferation, survival, and receptor acquisition To further define the kinetics of cytokines on NK differentiation from progenitors, UCB CD34+/Lin/CD38 cells were
cultured in direct contact with AFT024 ligands with FL, KL, and IL-3
alone, and with the addition of IL-7, IL-15, or IL-2 for 25 or 50 days.
At 25 days, the presence of NK cell differentiation was detected under
all conditions, including when FL, KL, and IL-3 were used alone in the
absence of a lymphocyte-stimulating cytokine, suggesting that NK cell
differentiation can occur on AFT024, albeit with low proliferation
(Table 2). These few NK cells that
differentiate without IL-7, IL-15, or IL-2 were predominantly CD94 (86% of cultures) and all were KIR.
However, by day 50, NK cells were no longer present in 72% of cultures
in the absence of IL-7, IL-15, or IL-2. IL-7 alone was sufficient to
induce acquisition of CD94 (75% to 89% of cultures positive) but
poorly supported KIR acquisition as shown by the 48% positive cultures
at day 25 and only 18% positive cultures at day 50. A majority
(> 70%) of cultures containing either IL-15 or IL-2 were positive
for both CD94 and at least one KIR at either time point.
There were distinct patterns of differentiation and proliferation of NK
cells between cytokine conditions (Figure
2A). At day 18, there were less than 500 absolute NK cells per 10 starting progenitors under all culture
conditions (data not shown). Culture of 10 UCB
CD34+/Lin
Single UCB CD34+/Lin /CD38 cells were
cultured from 2 donors with AFT024, FL, KL, IL-3, and single or
combinations of lymphocyte-stimulating cytokines. After about 50 days,
single cells were capable of NK cell differentiation and proliferation
similar to bulk cultures. The progeny of each single cell was then
analyzed for individual KIR, either KIR3DL1 (DX9, NKB1), KIR2DL1/S1
(EB6, CD158a), or KIR2DL2/L3/S2 (GL183, CD158b). All single UCB
progenitors resulted in NK cells with polyclonal KIR expression,
suggesting that KIR fate is determined at a maturational stage beyond a
CD34+/Lin/CD38 cell. Cultures
containing IL-2 + IL-7 resulted in the greatest number of
KIR-expressing NK cells (data not shown). Therefore, IL-7, IL-7 + IL-15, and IL-7 + IL-2 were studied further to determine the
frequency of individual KIR expression from single hematopoietic stem
cell populations.
Single UCB CD34+/Lin
NK cells express KIR irrespective of progenitor HLA type We designed experiments to address whether the HLA type of starting CD34+/Lin/CD38 cells
influenced KIR expression on developing NK cells (eg, would expression
of bw4 lead to more or less KIR3DL1 expression). The CD34
fraction of 6 UCB populations was serologically typed for class I MHC
to assign the presence or absence of known KIR ligands of corresponding
CD34+/Lin/CD38 cells. Single
progenitors were then sorted onto AFT024 and cytokines maximal for
inducing NK cell differentiation. Progeny were analyzed for KIR. For
these experiments, CD158a was detected by using HP-3E4, which stained
similar to EB6. Both recognize KIR2DL1/S1, but recently HP-3E4 has been
reported to also recognize KIR2DS4 (Assigned at Sixth International
Leukocyte Typing Workshop,
www.ncbi.nlm.nih.gov/prow/guide/682_373_991_g.htm). Twenty-nine
percent of single progenitors that were heterozygous or homozygous for
bw4 gave rise to NK cells expressing KIR3DL1. Similarly, of progenitors
that lacked bw4 (those that were homozygous bw6), 28% of NK cell
progeny expressed KIR3DL1. There was no difference in the absolute
number of KIR3DL1-expressing NK cells from single progenitors with or
without bw4. Similar results were seen when comparing starting
progenitors with or without a Cw4 or Cw3 group ligand (Table
4 and Figure 3I).
CD34+/Lin /CD38 cells were
sorted from fetal liver, UCB, and adult BM and cultured on AFT024 with
FL, KL, IL-3, IL-7, and IL-2 for 40 to 55 days. Proliferation of NK
cells was greatest with starting cells earlier in ontogeny (fetal
liver > UCB > adult BM). The absolute number of NK cells
derived from a single
CD34+/Lin/CD38 cell was
437 260 ± 54 721 from fetal liver (n = 26),
223 030 ± 20 589 from UCB (n = 144), and 15 584 ± 5923 from
adult BM (n = 59). Like UCB, fetal liver-derived NK cells expressed
KIR2DL2/L3/S2 with a significantly higher frequency than KIR2DL1/S1 or
KIR3DL1 (Figure 4C). The ratio of the average number of
GL183+ NK cells over the average number of DX9+
NK cells was 10.3 for fetal liver, 9.5 for UCB, and 1.9 for adult BM.
The preferential expression of KIR2DL2/L3/S2 on fetal liver-derived NK
cells was similar to that seen with UCB, and both were more pronounced
than with adult BM.
A novel murine fetal liver-derived stromal feeder (AFT024) was used to study NK cell differentiation and the ontogeny of human NK cell receptors. Using this feeder, CD94 and KIR acquisition were optimal when progenitors were in direct contact with AFT024 stromal ligands. Although receptor expression was dependent on exogenous cytokines, cytokines alone in the absence of AFT024 were inefficient in supporting NK differentiation and receptor expression. Soluble factors released by AFT024 only partially restored differentiation along the NK cell lineage and NK cell receptor acquisition. CD94 expression occurred earlier and at a greater frequency than KIR. Both were time dependent with few receptors expressed during short culture intervals. AFT024 supplemented with FL, KL, and IL-3 alone supported a small number of CD56+ NK cells, but these NK cells lacked CD94 and KIR and did not survive long term in the absence of a lymphocyte-stimulating cytokine. Direct comparisons between IL-7, IL-15, and IL-2 show that IL-7 alone is a weak inducer of CD94+ and KIR+ NK cells from primitive progenitors. IL-15 and IL-2 are equivalent at short culture intervals and IL-2 is significantly greater at long culture intervals. However, this does not imply that IL-2 has a different capacity to support NK cell differentiation or receptor acquisition but may only reflect the relative potency of these cytokines to induce NK cell proliferation from primitive progenitors in long-term culture. These bulk culture data agree with studies using a mixed CD34+ progenitor population cultured with FL followed by IL-15, resulting in NK cells that were mostly NKG2A+ and expressed some but significantly less KIR.43 The strength of this experimental system is its efficiency to study
single primitive cells. The AFT024 murine feeder is unique by
efficiently differentiating enough cells to analyze multiple receptors
and to calculate cloning efficiencies directly, results we were unable
to achieve with primary human stromal layers.40 In
addition, NK cells differentiated by AFT024 are similar to those
induced by primary human stroma, making this a good model to study NK
cell development. The capacity of murine stromal lines to support human
NK cell and B-cell differentiation has been reported by
others.46,54-56 From single
CD34+/Lin KIR acquisition was polyclonal and more than one receptor could be
expressed per cell. All 3 specific KIRs tested were represented in NK
cell progeny from single primitive progenitors. These results are
distinctly different from IL-15 containing stroma-free cultures using
thymic progenitors that differentiated into
CD94+/NKG2A+ NK cells but could not express KIR
as detected by immunofluorescence or reverse transcriptase-polymerase
chain reaction.57 Our data fit with the cumulative and
successive expression of Ly49 receptors in a stroma-dependent system in
the mouse.58 The capacity to express KIR in vitro
increased as the ontogeny of the stem cell source was more immature.
Fetal liver CD34+/Lin The expression of KIR did not correlate with the HLA type of the progenitor source. This was most definitively shown with class I typing for the mutually exclusive epitopes, Bw4 and Bw6. There was no difference in the number of single cells giving rise to KIR3DL1+ NK cell progeny or the absolute number of KIR3DL1+ NK cells from progenitors that were homozygous Bw6 (lacking Bw4) compared with progenitors containing at least one Bw4-containing allele. The same was true on the basis of serologic typing for the Cw group alleles. This finding agrees with those found in vivo according to a population study of more than 200 normal donors. Normal donors who are homozygous Bw6 still express KIR3DL1 on their circulating NK cells and there was no dose effect based on the presence of one or 2 Bw4 alleles, although studies of family members and twins showed some genetic linkage independent of HLA type.59 Taken together, our data and those published studies show that self alleles do not determine KIR acquisition. However, because all NK cells have at least one receptor recognizing self ligands, it is not yet known how class I alleles educate NK cells to ensure that appropriate receptors are selected. There has been more progress in the mouse to investigate what
determines NK cell receptor repertoires (reviewed in Raulet et
al60 and Hoglund et al61). Two major
hypotheses have been incorporated into models explaining the education
process of NK cells during development. The first hypothesis is that
each NK cell must have at least one self-specific NK cell inhibitory
receptor to silence auto-aggressive NK cells. The second presumption is that multiple self-specific NK cell receptors are disfavored by the
education process so that NK cells will maintain the ability to
discriminate between single allelic losses possibly induced by viral
infection or malignant transformation. Having too many NK inhibitory
receptors would decrease the sensitivity of NK cells to distinguish
these single allelic losses. Experiments using class I-deficient mice
have been instructive in supporting these hypotheses.62
Class I expression is not required for expression of NK cell inhibitory
receptors, and The control of NK cell receptor expression may be determined by
specific transcription factors that target NK cell receptor genes. Held
et al65 have shown that, although the circulating NK cell
compartment is normal in TCF-1 In summary, NK cell differentiation from single primitive human
progenitors requires contact with stroma and defined human cytokines to
induce CD94 and KIR on developing NK cells that is independent of
progenitor HLA type. The polyclonal nature of this expression shows
that receptor fate is determined beyond the maturational stage of an
uncommitted CD34+/Lin
We thank Brad Anderson for his help with flow cytometry, Dr Tucker LeBien for his helpful input and support, and Harriet Noreen for facilitating HLA typing and their interpretation.
Submitted January 29, 2001; accepted March 27, 2001.
Supported by grants R01-HL-55417 and PO1-CA-65493 from the National Institutes of Health and supported in part by grant M01-RR00400 from the National Cancer Institute for Research Resources.
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: Jeffrey S. Miller, University of Minnesota Cancer Center, Box 806, Division of Hematology, Oncology and Transplantation, Harvard Street at East River Road, Minneapolis, MN 55455; e-mail: mille011{at}tc.umn.edu.
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© 2001 by The American Society of Hematology.
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  M. J. J. Rose, A. G. Brooks, L. A. Stewart, T. H. Nguyen, and A. P. Schwarer Killer Ig-Like Receptor Ligand Mismatch Directs NK Cell Expansion In Vitro J. Immunol., October 1, 2009; 183(7): 4502 - 4508. [Abstract] [Full Text] [PDF]
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 S. Andersson, C. Fauriat, J.-A. Malmberg, H.-G. Ljunggren, and K.-J. Malmberg KIR acquisition probabilities are independent of self-HLA class I ligands and increase with cellular KIR expression Blood, July 2, 2009; 114(1): 95 - 104. [Abstract] [Full Text] [PDF]
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J. Yu, J. M. Venstrom, X.-R. Liu, J. Pring, R. S. Hasan, R. J. O'Reilly, and K. C. Hsu Breaking tolerance to self, circulating natural killer cells expressing inhibitory KIR for non-self HLA exhibit effector function after T cell-depleted allogeneic hematopoietic cell transplantation Blood, April 16, 2009; 113(16): 3875 - 3884. [Abstract] [Full Text] [PDF]
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B. Zal, J. C. Kaski, J. P. Akiyu, D. Cole, G. Arno, J. Poloniecki, A. Madrigal, A. Dodi, and C. Baboonian Differential Pathways Govern CD4+CD28- T Cell Proinflammatory and Effector Responses in Patients with Coronary Artery Disease J. Immunol., October 15, 2008; 181(8): 5233 - 5241. [Abstract] [Full Text] [PDF]
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M. Yawata, N. Yawata, M. Draghi, F. Partheniou, A.-M. Little, and P. Parham MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response Blood, September 15, 2008; 112(6): 2369 - 2380. [Abstract] [Full Text] [PDF]
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 C. Vitale, F. Cottalasso, E. Montaldo, L. Moretta, and M. C. Mingari Methylprednisolone induces preferential and rapid differentiation of CD34+ cord blood precursors toward NK cells Int. Immunol., April 1, 2008; 20(4): 565 - 575. [Abstract] [Full Text] [PDF]
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S. Santourlidis, N. Graffmann, J. Christ, and M. Uhrberg Lineage-Specific Transition of Histone Signatures in the Killer Cell Ig-Like Receptor Locus from Hematopoietic Progenitor to NK Cells J. Immunol., January 1, 2008; 180(1): 418 - 425. [Abstract] [Full Text] [PDF]
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H. Wang, B. Grzywacz, D. Sukovich, V. McCullar, Q. Cao, A. B. Lee, B. R. Blazar, D. N. Cornfield, J. S. Miller, and M. R. Verneris The unexpected effect of cyclosporin A on CD56+CD16 and CD56+CD16+ natural killer cell subpopulations Blood, September 1, 2007; 110(5): 1530 - 1539. [Abstract] [Full Text] [PDF]
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J. H. Chewning, C. N. Gudme, K. C. Hsu, A. Selvakumar, and B. Dupont KIR2DS1-Positive NK Cells Mediate Alloresponse against the C2 HLA-KIR Ligand Group In Vitro J. Immunol., July 15, 2007; 179(2): 854 - 868. [Abstract] [Full Text] [PDF]
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S. Cooley, F. Xiao, M. Pitt, M. Gleason, V. McCullar, T. L. Bergemann, K. L. McQueen, L. A. Guethlein, P. Parham, and J. S. Miller A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature Blood, July 15, 2007; 110(2): 578 - 586. [Abstract] [Full Text] [PDF]
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J. C. Fischer, H. Ottinger, S. Ferencik, M. Sribar, M. Punzel, D. W. Beelen, M. A. Schwan, H. Grosse-Wilde, P. Wernet, and M. Uhrberg Relevance of C1 and C2 Epitopes for Hemopoietic Stem Cell Transplantation: Role for Sequential Acquisition of HLA-C-Specific Inhibitory Killer Ig-Like Receptor J. Immunol., March 15, 2007; 178(6): 3918 - 3923. [Abstract] [Full Text] [PDF]
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B. Grzywacz, N. Kataria, M. Sikora, R. A. Oostendorp, E. A. Dzierzak, B. R. Blazar, J. S. Miller, and M. R. Verneris Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells Blood, December 1, 2006; 108(12): 3824 - 3833. [Abstract] [Full Text] [PDF]
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S. R. Presnell, L. Zhang, C. A. Ramilo, H.-W. Chan, and C. T. Lutz Functional redundancy of transcription factor-binding sites in the killer cell Ig-like receptor (KIR) gene promoter Int. Immunol., August 1, 2006; 18(8): 1221 - 1232. [Abstract] [Full Text] [PDF]
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S. L. Rogers, A. Rouhi, F. Takei, and D. L. Mager A Role for DNA Hypomethylation and Histone Acetylation in Maintaining Allele-Specific Expression of Mouse NKG2A in Developing and Mature NK Cells J. Immunol., July 1, 2006; 177(1): 414 - 421. [Abstract] [Full Text] [PDF]
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B. Giebel, T. Zhang, J. Beckmann, J. Spanholtz, P. Wernet, A. D. Ho, and M. Punzel Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division Blood, March 1, 2006; 107(5): 2146 - 2152. [Abstract] [Full Text] [PDF]
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S. Cooley, V. McCullar, R. Wangen, T. L. Bergemann, S. Spellman, D. J. Weisdorf, and J. S. Miller KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation Blood, December 15, 2005; 106(13): 4370 - 4376. [Abstract] [Full Text] [PDF]
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C.-W. Lin, T.-Y. Liu, S.-U. Chen, K.-T. Wang, L. J. Medeiros, and S.-M. Hsu CD94 1A transcripts characterize lymphoblastic lymphoma/leukemia of immature natural killer cell origin with distinct clinical features Blood, November 15, 2005; 106(10): 3567 - 3574. [Abstract] [Full Text] [PDF]
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H.-W. Chan, J. S. Miller, M. B. Moore, and C. T. Lutz Epigenetic Control of Highly Homologous Killer Ig-Like Receptor Gene Alleles J. Immunol., November 1, 2005; 175(9): 5966 - 5974. [Abstract] [Full Text] [PDF]
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P. S. Woll, C. H. Martin, J. S. Miller, and D. S. Kaufman Human Embryonic Stem Cell-Derived NK Cells Acquire Functional Receptors and Cytolytic Activity J. Immunol., October 15, 2005; 175(8): 5095 - 5103. [Abstract] [Full Text] [PDF]
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M. I. Lutskiy, D. S. Beardsley, F. S. Rosen, and E. Remold-O'Donnell Mosaicism of NK cells in a patient with Wiskott-Aldrich syndrome Blood, October 15, 2005; 106(8): 2815 - 2817. [Abstract] [Full Text] [PDF]
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 J. Xu, A. N. Vallejo, Y. Jiang, C. M. Weyand, and J. J. Goronzy Distinct Transcriptional Control Mechanisms of Killer Immunoglobulin-like Receptors in Natural Killer (NK) and in T Cells J. Biol. Chem., June 24, 2005; 280(25): 24277 - 24285. [Abstract] [Full Text] [PDF]
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J. S. Miller, Y. Soignier, A. Panoskaltsis-Mortari, S. A. McNearney, G. H. Yun, S. K. Fautsch, D. McKenna, C. Le, T. E. Defor, L. J. Burns, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer Blood, April 15, 2005; 105(8): 3051 - 3057. [Abstract] [Full Text] [PDF]
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G. Katz, R. Gazit, T. I. Arnon, T. Gonen-Gross, G. Tarcic, G. Markel, R. Gruda, H. Achdout, O. Drize, S. Merims, et al. MHC Class I-Independent Recognition of NK-Activating Receptor KIR2DS4 J. Immunol., August 1, 2004; 173(3): 1819 - 1825. [Abstract] [Full Text] [PDF]
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L. D. Lieto, F. Borrego, C.-h. You, and J. E. Coligan Human CD94 Gene Expression: Dual Promoters Differing in Responsiveness to IL-2 or IL-15 J. Immunol., November 15, 2003; 171(10): 5277 - 5286. [Abstract] [Full Text] [PDF]
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C.-W. Lin, Y.-H. Chen, Y.-C. Chuang, T.-Y. Liu, and S.-M. Hsu CD94 transcripts imply a better prognosis in nasal-type extranodal NK/T-cell lymphoma Blood, October 1, 2003; 102(7): 2623 - 2631. [Abstract] [Full Text] [PDF]
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S. Rutella, G. Bonanno, M. Marone, D. de Ritis, A. Mariotti, M. T. Voso, G. Scambia, S. Mancuso, G. Leone, and L. Pierelli Identification of a Novel Subpopulation of Human Cord Blood CD34-CD133-CD7-CD45+Lineage- Cells Capable of Lymphoid/NK Cell Differentiation After In Vitro Exposure to IL-15 J. Immunol., September 15, 2003; 171(6): 2977 - 2988. [Abstract] [Full Text] [PDF]
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I. Barao, D. Hudig, and J. L. Ascensao IL-15-Mediated Induction of LFA-1 Is a Late Step Required for Cytotoxic Differentiation of Human NK Cells from CD34+Lin- Bone Marrow Cells J. Immunol., July 15, 2003; 171(2): 683 - 690. [Abstract] [Full Text] [PDF]
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V. Ioannidis, B. Kunz, D. M. Tanamachi, L. Scarpellino, and W. Held Initiation and Limitation of Ly-49A NK Cell Receptor Acquisition by T Cell Factor-1 J. Immunol., July 15, 2003; 171(2): 769 - 775. [Abstract] [Full Text] [PDF]
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E. G. Chiorean, S. J. Dylla, K. Olsen, T. Lenvik, Y. Soignier, and J. S. Miller BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIRs) Blood, May 1, 2003; 101(9): 3527 - 3533. [Abstract] [Full Text] [PDF]
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H. G. Shilling, K. L. McQueen, N. W. Cheng, J. A. Shizuru, R. S. Negrin, and P. Parham Reconstitution of NK cell receptor repertoire following HLA-matched hematopoietic cell transplantation Blood, May 1, 2003; 101(9): 3730 - 3740. [Abstract] [Full Text] [PDF]
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 H.-W. Chan, Z. B. Kurago, C. A. Stewart, M. J. Wilson, M. P. Martin, B. E. Mace, M. Carrington, J. Trowsdale, and C. T. Lutz DNA Methylation Maintains Allele-specific KIR Gene Expression in Human Natural Killer Cells J. Exp. Med., January 20, 2003; 197(2): 245 - 255. [Abstract] [Full Text] [PDF]
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S. S. Farag, T. A. Fehniger, L. Ruggeri, A. Velardi, and M. A. Caligiuri Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect Blood, August 28, 2002; 100(6): 1935 - 1947. [Abstract] [Full Text] [PDF]
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H. G. Shilling, N. Young, L. A. Guethlein, N. W. Cheng, C. M. Gardiner, D. Tyan, and P. Parham Genetic Control of Human NK Cell Repertoire J. Immunol., July 1, 2002; 169(1): 239 - 247. [Abstract] [Full Text] [PDF]
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M. R. Snyder, L.-O. Muegge, C. Offord, W. M. O'Fallon, Z. Bajzer, C. M. Weyand, and J. J. Goronzy Formation of the Killer Ig-Like Receptor Repertoire on CD4+CD28null T Cells J. Immunol., April 15, 2002; 168(8): 3839 - 3846. [Abstract] [Full Text] [PDF]
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 S. Sivori, M. Falco, E. Marcenaro, S. Parolini, R. Biassoni, C. Bottino, L. Moretta, and A. Moretta Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation PNAS, March 21, 2002; (2002) 72065999. [Abstract] [Full Text] [PDF]
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S. Sivori, M. Falco, E. Marcenaro, S. Parolini, R. Biassoni, C. Bottino, L. Moretta, and A. Moretta Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation PNAS, April 2, 2002; 99(7): 4526 - 4531. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
exactly the opposite. As originally proposed by
Ljunggren and Karre
(CD122) expression
on NK cell progenitors.
), separated
from the feeder by a Transwell membrane (
), or in cytokines alone in
the absence of AFT024 (
). All cultures were supplemented with weekly
FL in combination with KL alone, or with the addition of a
lymphocyte-stimulating cytokine (IL-7, IL-15, or IL-2). IL-3 was only
added once at culture initiation. After 30 to 34 days, cell progeny
were harvested and evaluated for the absolute number of
CD56+, CD56+/CD94+, and
CD56+/KIR+ NK cells. (A) The absolute number of
NK cells was greatest in the presence of IL-15 or IL-2 when progenitors
were cultured in direct contact with AFT024. Under this condition, NK
cells accounted for more overall cell growth after the addition of
IL-15 (49% ± 5.6%) or IL-2 (56% ± 7.3%) compared with the
addition of IL-7 (1.6% ± 0.6%) or in the absence of a
lymphocyte-stimulating cytokine (0.6% ± 0.2%). (B) The absolute
number of NK cells expressing CD94 (clone HP-3B1) accounted for
38% ± 9.1% of total NK cells for IL-15 and 49% ± 8.5% for
IL-2 cultures in direct contact with AFT024. (C) The absolute number of
NK cells expressing at least one of the known KIRs (using a PE-cocktail
of 3 monoclonal antibodies, DX9, GL183, and EB6) accounted for
2.8% ± 0.8% of total NK cells for IL-15 and 4.9% ± 0.8% for
IL-2 cultures in direct contact with AFT024. There were no (FL, KL,
IL-3 ± IL-7) or few (+IL-15 or +IL-2) KIR-expressing NK cells in
cultures containing cytokines alone in the absence of AFT024.
P values are listed for significant differences between
progenitors separated from stroma or with cytokines alone compared with
NK cell differentiation in contact with AFT024. Each bar represents the
mean ± SEM from 6 to 9 UCB progenitor populations sorted for each
condition. The insert to the right of the graph shows IL-7 data with a
smaller scale to show differences.
KIR3DL1) with either cytokine combination. Although EB6 and DX9 staining were similar in some individual experiments, GL183
always detected a higher percentage of single cell-derived NK progeny
than EB6 or DX9. A majority of KIR+ NK cells also expressed
CD94 (98% ± 0.2% for IL-2- [n = 17] and 99% ± 0.5% for
IL-15-containing [n = 111] cultures) but rare
KIR+/CD94
, adult marrow
(n = 59).
-helical mutations that affect T cells.
J Immunol.
1995;154:2631-2641