Download Are Targeted by NK Cells Hematopoietic Progenitors Express H60

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HOXB4-Transduced Embryonic Stem
Cell-Derived Lin−c-kit+ and Lin−Sca-1+
Hematopoietic Progenitors Express H60 and
Are Targeted by NK Cells
William B. Tabayoyong, Juan G. Salas, Sabrina Bonde and
Nicholas Zavazava
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Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:5449-5457; Prepublished online 14
October 2009;
doi: 10.4049/jimmunol.0901807
http://www.jimmunol.org/content/183/9/5449
The Journal of Immunology
HOXB4-Transduced Embryonic Stem Cell-Derived Linⴚc-kitⴙ
and LinⴚSca-1ⴙ Hematopoietic Progenitors Express H60 and
Are Targeted by NK Cells1
William B. Tabayoyong,* Juan G. Salas,† Sabrina Bonde,† and Nicholas Zavazava2*†
E
mbryonic stem (ES)3 cells have emerged as a promising
new cell source for cell replacement therapies because of
their ability to develop into cell types derived from all
three embryonal germ layers (1). Furthermore, ES cells possess a
high proliferative capacity and can be propagated indefinitely in
culture without loss of pluripotency (2). To date, several protocols
are available to develop ES cells into neurons (3), cardiomyocytes
(4), endothelial cells (5), and hematopoietic precursors (6 – 8).
However, a major challenge to the therapeutic application of ES
cell derivatives is their potential for recognition by the immune
system.
The first reports on ES cell immunogenicity demonstrated that
undifferentiated ES cells and differentiated cells derived from the
embryoid body (EB) expressed low levels of MHC class I and no
class II Ags (9 –11). Additionally, ES cells and their EB derivatives were not susceptible to NK cell cytotoxicity and they failed
to stimulate alloreactive T cell proliferation in both murine and
human cells in vitro (9 –11). These findings led to the belief that ES
cells are immune privileged and can be readily transplanted across
MHC barriers with minimal immunosuppression.
In support of this idea, in vivo studies using a humanized mouse
model to characterize the immunogenicity of human ES cells and
EB cells demonstrated that human ES cells and their EB derivatives induced considerably weaker allogeneic immune responses
*Medical Scientist Training Program and Immunology Graduate Program, University
of Iowa, Iowa City, Iowa 52242; and †Department of Internal Medicine, University of
Iowa and Veteran Affairs Medical Center, Iowa City, Iowa 52246
Received for publication June 8, 2009. Accepted for publication September 3, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health R01 HL073015 and
Veteran Affairs Merit Review.
than adult allografts (12). Furthermore, studies from our own laboratory demonstrated that undifferentiated mouse ES cells were
nonimmunogenic and capable of populating both primary and secondary lymphoid organs in sublethally irradiated recipients (13).
In contrast, other in vivo studies have challenged the idea of ES
cell immune privilege. For example, Robertson et al. (14) reported
that murine EBs transplanted under the kidney capsule of recipient
mice were fully immunogenic and succumbed to rejection by an
allogeneic immune response. Moreover, two studies evaluating the
potential of ES cells to treat myocardial infarction demonstrated
that undifferentiated ES cells transplanted into ischemic myocardium were rejected by graft-infiltrating T cells and induced an
alloantibody humoral response (15, 16).
Currently, the reasons for the discrepancy observed in these
studies are unclear. It is possible that inflammation post transplantation could have led to the up-regulation of MHC Ags on ES cells
and increased their immunogenicity. Indeed, IFN-␥, a key inflammatory cytokine, has been shown to increase the expression of
MHC class I on ES and EB cells (10, 12, 13). However, it is
important to note that both undifferentiated ES and EB cells are
heterogeneous cell populations and are not suitable for immunological studies. Therefore, studies on purified, terminally differentiated cell types derived from ES cells are required to allow full
characterization of the immune response against ES-derived cells.
In this study, we examined the impact of NK cells on the longterm engraftment of purified CD45⫹ ES cell-derived hematopoietic progenitor cells (HPC) by using NK-deficient Rag2⫺/⫺␥c⫺/⫺
and NK-replete Rag2⫺/⫺ mice. We hypothesized that NK cells are
a major immunological barrier to HPC engraftment due to low
MHC expression by HPCs. We demonstrate that ES cell-derived
HPCs express high levels of H60, a well-characterized ligand for
the NK activating receptor NKG2D (17–20), rendering HPCs susceptible to NK killing in vitro and in vivo.
2
Address correspondence and reprint requests to Dr. Nicholas Zavazava, University
of Iowa Hospitals and Clinics, Department of Internal Medicine, 200 Hawkins Drive,
C42 E6 GH, Iowa City, Iowa 52242. E-mail address: [email protected]
Materials and Methods
ES cell lines and culture conditions
3
Abbreviations used in this paper: ES cell, embryonic stem cell; EB, embryoid body;
HPC, hematopoietic progenitor cell.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901807
The HOXB4-transduced CCE ES cell line (129 SvJ origin, H-2b), provided
by Dr. Hannes Klump (University of Essen, Essen, Germany) (7), was
grown on gelatinized flasks in feeder cell-free ES cell culture medium
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Embryonic stem (ES) cells are a novel source of cells, especially hematopoietic progenitor cells that can be used to treat degenerative diseases in humans. However, there is a need to determine how ES cell-derived progenitors are regulated by both the
adaptive and innate immune systems post transplantation. In this study, we demonstrate that hematopoietic progenitor cells
(HPCs) derived from mouse ES cells ectopically expressing HOXB4 fail to engraft long-term in the presence of NK cells. In
particular, the H60-expressing Linⴚc-kitⴙ and LinⴚSca-1ⴙ subpopulations were preferentially deleted in Rag2ⴚ/ⴚ, but not in
Rag2ⴚ/ⴚ␥cⴚ/ⴚ mice. Up-regulation of class I expression on HPCs prevented their lysis by NK cells, and Ab-mediated depletion of
NK cells restored long-term HPC engraftment. In contrast to the notion that ES-derived cells are immune-privileged, we show in
this study that NK cells form a formidable barrier to the long-term engraftment of ES cell-derived hematopoietic
progenitors. The Journal of Immunology, 2009, 183: 5449 –5457.
5450
NK CELLS TARGET ES CELL-DERIVED HEMATOPOIETIC CELLS
consisting of DMEM (Life Technologies/BRL) supplemented with 15%
FCS, 0.1 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and
1000 U/ml leukemia inhibitory factor. Culture medium was changed daily,
and the cells were passaged every 2–3 days to avoid overgrowth and
differentiation.
In vitro generation of ES cell-derived HPCs
ES cells were differentiated into HPCs as we and colleagues previously
described (7, 8). In brief, ES cells were subjected to EB formation. EBs
were dissociated into a single-cell suspension and replated onto ultra-low
attachment Petri dishes in serum-free hematopoietic differentiation medium
containing StemPro34 plus nutrient supplement (Life Technologies/BRL)
and a mixture of hematopoietic cytokines including mouse stem cell factor
(100 ng/ml, R&D Systems), mIL-3 (2 ng/ml), mIL-6 (5 ng/ml), Flt3-L (10
ng/ml), IGF-1 (40 ng/ml, Promega), and dexamethasone (1 ␮M, SigmaAldrich). Culture medium was changed every day and cell density was
maintained below 4 ⫻ 106 cells/ml for an additional 20 days. At this point,
70 –96% of the cells were CD45⫹ (8). To remove nondifferentiated cells,
CD45⫹ HPCs were positively selected using immunomagnetic beads
(Miltenyi Biotec) according to the manufacturer’s recommended protocol.
Mice and cell transplantation
Flow cytometry
Cells were stained with fluorochrome-conjugated Abs against H-2Kb
(AF6 – 88.5), Qa-2 (1–1-2), IA/IE (M5/114.15.2), c-kit (2B8), Sca-1 (E13–
161.7), CD3 (17A2), CD11c (HL3), Mac-1 (M1/70), Gr-1 (RB6 – 8C5),
CD16 (2.4G2), CD62L (MEL-14), and CD69 (H1.2F3), purchased from
BD Biosciences; NK1.1 (PK136), NKG2D (CX5), and CD25 (PC61.5),
purchased from eBioscience; B220 (RA3– 6B2), CD49b (DX5), and CD44
(IM7), purchased from BioLegend. Abs against Rae-1 (199205), MULT-1
(237104), and H60 (205326) were purchased from R&D Systems. Data
were acquired on a BD FACScan or LSR II and analyzed with Cell Quest
Software.
Immunoblotting
To analyze granzyme B and perforin expression in NK cells, protein lysates
were generated from splenocytes harvested from naive Rag2⫺/⫺ mice and
Rag2⫺/⫺ mice that had received either an i.p. injection of 200 ␮g poly I:C
or an i.v. injection of 107 HPCs 24 h before the time of harvest. Lysates
were also generated from splenocytes harvested from Rag2⫺/⫺ mice receiving both an i.p. injection of 200 ␮g poly I:C and an i.v. injection of 107
HPCs 24 h before harvest. Reducing SDS-PAGE, transfer to polyvinylidene fluoride membranes, and immunoblots for perforin, granzyme B, and
actin were performed as described (22), with 20 ␮g total protein run in each
lane. Rat anti-mouse perforin (P1– 8, Kamiya, 1/5000) and rat anti-mouse
granzyme B (16G6, eBioscience, 1/1000) were used and developed with a
goat anti-rat IgG HRP conjugated secondary Ab (Santa Cruz Biotechnology). HRP-conjugated mouse anti-mouse ␤-actin (C4, Santa Cruz Biotechnology, 1/5000) was used as a loading control. Samples were detected
using the ECL Plus Western blotting detection system (Amersham Biosciences) per the manufacturer’s instructions.
IFN-␥ ELISPOT
To analyze IFN-␥ production by NK cells, splenocytes were harvested
from naive Rag2⫺/⫺ mice or Rag2⫺/⫺ mice that had received an i.v. injection of 107 HPCs 6 h before the time of harvest. This time was chosen
because our prior experiments had shown that NK cell activity peaked at
this time. Splenocytes were then enriched for NK cells by negative selection (Miltenyi Biotec). Five ⫻ 105 naive NK cells or NK cells from HPCtransplanted mice were then cultured in duplicate with 5 ⫻ 105 HPCs or 10
ng/ml PMA and 1 ␮g/ml ionomycin overnight, and IFN-␥ production was
determined using the IFN-␥ enzyme linked immunospot assay (BD Biosciences) according to the manufacturer’s instructions.
Depletion of NK1.1⫹ cells
NK cells in Rag2⫺/⫺ recipient mice were depleted as described (23). In
brief, Rag2⫺/⫺ mice were injected i.p. with 200 ␮g anti-NK1.1 mAb
IFN-␥ treatment and in vivo NK cell cytotoxicity
To up-regulate HPC expression of MHC class I, HPCs were treated in vitro
with IFN-␥ (PeproTech) at a final concentration of 20 ng/ml for 72 h. To
confirm up-regulation of MHC Ags, MHC class I and II expression were
measured by flow cytometry at 24, 48, and 72 h. The in vivo NK cell
cytotoxicity assay was adapted from previously described T and NK cell
killing assays (24 –26) using HPCs treated with IFN-␥ for 48 h and nontreated HPCs as target cells adoptively transferred into Rag2⫺/⫺ and
Rag2⫺/⫺␥c⫺/⫺ mice. The Rag2⫺/⫺␥c⫺/⫺ mice served as the negative control for NK killing. To discriminate the IFN-␥-treated HPCs that had upregulated MHC class I expression from the nontreated HPC pool, the
IFN-␥ treated HPCs were labeled with the red membrane dye PKH26 per
the manufacturer’s protocol (Sigma-Aldrich). A mixture of 107 PKH26labeled IFN-␥-treated HPCs and 107 PKH26-unlabeled HPCs was then
adoptively transferred by i.v. injection into recipient mice. Splenocytes
were harvested from recipient animals at 1, 6, and 24 h after adoptive
transfer, and analyzed by flow cytometry. Additionally, 24 h post adoptive
transfer, bone marrow cells were harvested and analyzed by flow cytometry
for the expression of c-kit, Sca-1, and the lineage markers CD3, B220,
CD11c, Mac-1, and Gr-1. The entire target cell pool was first distinguished
from the recipients’ endogenous splenocytes or bone marrow cells on the
basis of GFP expression, then the IFN-␥ treated HPCs were further discriminated from the nontreated HPCs on the basis of PKH26 expression. At
least two million events were acquired for each sample to ensure adequate
analysis of the adoptively transferred target cell populations. Percent killing was calculated using the following formula: {1⫺[((%HPC
remaining)(%IFN ␥ TreatedHPCtransplanted))/((%IFN ␥ TreatedHPC
remaining)(%HPCtransplanted))]}⫻100%.
Flow-based cytotoxicity assay
To measure in vitro cellular cytotoxicity of NK cells against HPCs, a previously described flow-based cytotoxicity assay (22, 27) was adapted. In
brief, GFP⫹ HPCs were plated in duplicate in 96-well U-bottom plates
with effector splenocytes harvested from polyI:C treated Rag2⫺/⫺ mice at
an E:T ratio of 20 to 1, and incubated at 37°C and 5% CO2 for 8 h.
Immediately before analysis, Hoechst 33258 (Invitrogen) was added to
each sample at a final concentration of 4 ␮g/ml. Hoechst 33258 incorporation served as a surrogate marker for late cell death/apoptosis, intercalating with DNA in cells that have lost membrane integrity. To analyze the
role of H60 and Rae-1 in NK cell killing of HPCs, blocking Abs against
H60 (205326) and Rae-1 (199205) or isotype control were added at the
indicated concentrations. Percent inhibition was calculated using the following formula: [1 – (% lysis with Ab/% lysis without antibody)] ⫻ 100.
Statistical analysis
The Prism software package (GraphPad Software) was used to apply the
Students t test to evaluate experimental data for significant differences; p ⬍
0.05 was considered significant.
Results
HPCs express the NK-activating ligand H60
NK cells, effector lymphocytes of the innate immune system, are
cytotoxic to aberrant cells such as tumor cells or virally infected
cells that have down-regulated the expression of MHC class I Ags
(28). Several studies have documented low levels of MHC expression by ES cell-derived cells (10, 11, 13). Although low level
MHC expression is advantageous for avoiding recognition and rejection by T- and B cell-mediated immune responses, it could increase susceptibility to NK cell-mediated rejection. In this study,
we hypothesized that ES cell-derived HPCs are susceptible to NK
killing. First, we analyzed HPCs derived from the CCE ES cell line
transduced with HOXB4 by flow cytometry for the expression of
NKG2D activating ligands Rae-1, MULT-1, and H60, and the
MHC class I inhibitory ligands H-2Kb and Qa-2 (29). The differentiation of ES cells into HPCs resulted in significant up-regulation of H60 expression and modest increases in H-2Kb and Qa-2.
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Six- to eight-week-old Rag2⫺/⫺ and Rag2⫺/⫺␥c⫺/⫺ mice on the B6/B10
mixed inbred background were purchased from Taconic Farms (21). All
mice were housed in the animal facility at the Veteran Affairs Medical
Center, Iowa. All animal procedures were previously approved by the Institutional Animal Care and Use Committee (IACUC) and in accordance
with National Institutes of Health guidelines. HPCs were transplanted i.v.
as previously described (13).
(PK136, eBioscience) on days ⫺3 and ⫺1 before HPC transplantation.
Another group of Rag2⫺/⫺ recipient mice was similarly treated with a
mouse IgG2a, ␬ isotype control mAb (eBM2a, eBioscience) as a negative
control. To confirm depletion, peripheral blood was drawn from antiNK1.1 or isotype control treated mice on day 0 and stained with antiCD49b mAb (DX5, BioLegend) and analyzed by flow cytometry.
The Journal of Immunology
5451
The expression of all other molecules analyzed remained low (Fig.
1). To confirm that ectopic expression of HOXB4 did not influence
HPC expression of NK ligands, ES cells were similarly differentiated into HPCs and their expression of NK ligands measured by
flow cytometry. As expected, the expression of all molecules analyzed on the non-HOXB4 HPCs was similar to that of HOXB4transduced HPCs (Fig. 1), indicating that ectopic expression of
HOXB4 did not alter the expression of NK ligands in ES
cell-derived HPCs.
HPCs are poorly sensitive to NK killing in vitro but not in vivo
To determine whether NK cells lyse HPCs, we performed an in
vitro flow-based cytotoxicity assay. We observed that NK cells
could indeed kill HPCs, consistent with the HPCs’ NK stimulatory
phenotype observed in Fig. 1, albeit at only 25% efficiency relative
to Yac-1 positive control target cells (Fig. 2A). Again, the presence
of HOXB4 did not alter the susceptibility of HPCs to NK cellmediated killing because non-HOXB4 HPCs were lysed at similar
levels. To further examine whether HPC susceptibility could be
generalized to cells derived from unrelated ES cell lines, HPCs
derived from HOXB4-transduced BALB/c ⫻ 129SvJ F1 ES cells
were tested as target cells in the same assay. Cell killing was the
same as that of HPCs derived from CCE ES cells. These data
demonstrate that HOXB4 neither influenced the phenotype nor the
susceptibility of ES cell-derived HPCs to NK cell killing; therefore, the remaining experiments presented in this study were performed with HOXB4-derived HPCs. We have reported that
HOXB4-transduced HPCs stably engraft in lethally irradiated recipient mice while non-HOXB4-transduced HPCs lack self-renewal properties and fail to engraft (8, 13). Thus, the use of
HOXB4-transduced HPCs was critical because it allowed us to
distinguish between engraftment failure and NK cell-mediated
rejection.
Next, to determine whether NK cells kill HPCs in vivo, we used
the Rag2⫺/⫺ and the NK-cell deficient Rag2⫺/⫺␥c⫺/⫺ mice as
recipient mice of differentiated and purified CD45⫹ HPCs. As we
previously described, these HPCs were engineered to express GFP,
allowing easy monitoring of engrafted cells by flow cytometry (8).
Mice were lethally irradiated, and each subsequently transplanted
with 3 ⫻ 106 HPCs and 5 ⫻ 105 autologous bone marrow cells. On
day 14, 67% of PBMC in the Rag2⫺/⫺␥c⫺/⫺ recipient mice were
HPC-derived compared with only 14% in Rag2⫺/⫺ recipient mice
(Fig. 2B), suggesting regulation of engraftment by NK cells as the
Rag2⫺/⫺ mice have neither T nor B cells. Curiously, not all HPCs
were deleted in these mice, but rather a small population remained
detectable, suggesting that this subpopulation was protected from
deletion by NK cells. This NK cell-resistant subpopulation potentially consists of mature hematopoietic cells expressing MHC class
I molecules, consistent with our previously published immunophenotyping data that showed heterogeneity within the total HPC population (8). Indeed, HPC engraftment partially recovered in
Rag2⫺/⫺ recipients by day 28, with the GFP⫹ HPCs detectable in
peripheral blood rebounding to ⬃40%. Interestingly, by day 70,
HPC derived hematopoietic cells had decreased to ⬍3% in
Rag2⫺/⫺ mice compared with ⬎80% in Rag2⫺/⫺␥c⫺/⫺ recipients,
which remained stable over 100 days (Fig. 2C). These results are
consistent with our recently published data on HPC engraftment in
immunocompetent mice (30). We therefore concluded that NK
cells regulate HPC engraftment early after transplant, consistent
with the reported rapid kinetics of NK cell-mediated rejection of
transplanted allogeneic bone marrow cells (26, 31). To rule out
that the effects of lethal irradiation were confounding our engraftment data, we characterized both the percentage and absolute numbers of NK cells in peripheral blood, spleen, and bone marrow in
nonirradiated and irradiated Rag2⫺/⫺ mice. In irradiated mice, we
observed a decreased percentage of peripheral blood NK cells and
increased percentage of splenic NK cells; however, these differences did not reach statistical significance (supplementary Table
I).4 Absolute NK cell counts in the spleen after irradiation were
4
The online version of this article contains supplementary material.
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FIGURE 1. ES cell and HPC expression of NK activating and inhibitory ligands. Flow cytometric analysis of undifferentiated ES cells, HPCs derived
from HOXB4 transduced ES cells, and non-HOXB4 HPCs comparing the expression of Rae-1, MULT-1, H60, H-2Kb, and Qa-2.
5452
NK CELLS TARGET ES CELL-DERIVED HEMATOPOIETIC CELLS
slightly increased, while those in the bone marrow were slightly
decreased (supplementary Table II), yet, these differences were not
statistically significant. Furthermore, the ability of NK cells from
irradiated Rag2⫺/⫺ mice to kill Yac-1 target cells in an in vitro
flow-based cytotoxicity assay was similar to that of NK cells from
nonirradiated Rag2⫺/⫺ mice (supplementary Fig. 1); therefore, we
concluded that lethal irradiation did not influence the NK cell response against ES-derived HPCs.
HPC transplantation activates recipient NK cells
If indeed NK cells delete HPCs, we reasoned that it should be
possible to detect NK cell activation post transplant. Resting murine NK cells are minimally cytotoxic due to a block in perforin
and granzyme B mRNA translation, resulting in low perforin and
granzyme B protein expression (22). This block is released upon
NK cell activation by cytokines in vitro, poly I:C treatment, or
viral infection in vivo (22). To determine whether HPCs activated
NK cells in vivo, we transplanted Rag2⫺/⫺ mice with HPCs.
Splenocytes of transplanted Rag2⫺/⫺ mice were harvested after
24 h and perforin and granzyme B expression were analyzed by
Western blotting. Resting splenocytes harvested from naive
Rag2⫺/⫺ mice or activated splenocytes harvested from poly I:Ctreated Rag2⫺/⫺ mice served as negative and positive controls,
respectively. As expected, poly I:C treatment augmented the expression of perforin and granzyme B compared with naive animals,
with considerably higher levels of granzyme B detected than perforin (Fig. 3A). HPC-transplanted Rag2⫺/⫺ mice showed an upregulation of perforin expression that was not observed in controls,
suggesting that HPC transplantation activates NK cells. However,
the amount of granzyme B detected in HPC transplanted Rag2⫺/⫺
mice was less than that observed for poly I:C treated mice. We
speculated that this difference could be due to the presence of
target cells that trigger the release of granzyme B and perforin.
Thus, to determine whether HPC target cells led to the degranulation of NK cells, an additional Western blot for granzyme B
was performed using protein lysates isolated from splenocytes of
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FIGURE 2. NK cells regulate HPC engraftment in vivo. A, A flowbased cytotoxicity assay was performed using splenocytes derived from
polyI:C-treated Rag2⫺/⫺ mice as the source of NK effectors against
HOXB4-HPCs (CCE HOXB4), non-HOXB4-HPCs (CCE), or HOXB4HPCs derived from BALB/c ⫻ 129SvJ F1 ES cells (F1 HOXB4) at an E:T
ratio of 20 to 1. Percent lysis was calculated relative to positive control
Yac-1 target cells, with the median lysis of Yac-1 target cells set at 100%.
B, HPCs were transplanted into either Rag2⫺/⫺␥c⫺/⫺ or Rag2⫺/⫺ mice and
the percentage of HPC-derived cells in peripheral blood was monitored by
measuring GFP expression on day 14. Representative flow plots from individual Rag2⫺/⫺␥c⫺/⫺ and Rag2⫺/⫺ recipient mice are shown. C, Summary of the percentage of HPC-derived cells in the peripheral blood of
Rag2⫺/⫺␥c⫺/⫺ (n ⫽ 4) and Rag2⫺/⫺ (n ⫽ 4) recipient mice up to day 100
post transplant. Data are represented as mean ⫾ SEM; ⴱ, p ⬍ 0.05.
FIGURE 3. HPC transplantation activates recipient NK cells. A, Immunoblots of splenocytes for perforin, granzyme B, and actin. Protein lysates
were generated from splenocytes harvested from naive Rag2⫺/⫺ mice
(Resting) or Rag2⫺/⫺ mice that were either treated with 200 ␮g poly I:C or
transplanted with 107 HPCs 24 h before the time of harvest. B, Protein
lysates were generated from splenocytes harvested from Rag2⫺/⫺ mice
receiving both poly I:C treatment and HPC transplantation 24 h before
harvest. Lysates were analyzed for the expression of granzyme B. Actin
was used as a loading control. Results shown are representative of two
independent experiments. C, IFN-␥ production by NK cells harvested from
naive Rag2⫺/⫺ mice or activated NK cells harvested from Rag2⫺/⫺ mice
receiving HPC transplant. NK cells were stimulated overnight by either
coculture with HPCs or PMA/Ionomycin and IFN-␥ was measured by
ELISPOT. Results shown are representative of two independent experiments. Data are represented as mean ⫾ SEM; ⴱ, p ⬍ 0.05.
The Journal of Immunology
5453
NK cell depletion augments HPC engraftment
To confirm that NK cells regulate HPC engraftment, we depleted
NK cells in Rag2⫺/⫺ recipients using an anti-NK1.1 mAb. This Ab
effectively reduced the percentage of NK cells detected in peripheral blood from 40% to less than 5% (Fig. 4A). Anti-NK1.1- or
isotype control mAb-treated Rag2⫺/⫺ mice were lethally irradiated
and transplanted with HPCs. As early as day 7 post transplant, the
effect of NK cells on limiting HPC engraftment was evident. Only
2% of peripheral blood cells were GFP expressing in isotype control mAb treated Rag2⫺/⫺ recipients compared with 13% in NKdepleted Rag2⫺/⫺ recipients, suggesting that NK cell depletion
indeed augments HPC engraftment (Fig. 4B). Enhanced engraftment was most evident on day 28 post transplant; however, in
long-term studies the percentage of HPC-derived cells contracted
by day 70 (Fig. 4C), suggesting a re-emergence of NK cells because recipients were only treated with two doses of the depleting
Ab at the time of transplantation. Repetitive depletion of NK cells
may be warranted to allow permanent HPC engraftment. These
data confirm that NK cells regulate HPC engraftment and may
pose a previously unrecognized barrier for the transplantation of
ES-cell derivatives.
MHC class I expression protects HPCs from NK cell
cytotoxicity in vivo
We next sought to determine the mechanism by which the surviving HPCs evade NK cell-mediated cytotoxicity. Because MHC
class I molecules are the dominant inhibitory NK cell ligands, we
hypothesized that the HPCs that were protected from NK cell killing expressed class I Ags. To test this hypothesis, we deliberately
enhanced the expression of MHC class I Ags on HPCs by IFN-␥
stimulation. Consistent with previous reports (10, 13, 32), this
stimulation significantly increased MHC class I expression on
HPCs, but had no effect on MHC class II expression (Fig. 5A).
MHC class I expression peaked after 48 h of IFN-␥ treatment, with
⬎95% of HPCs expressing MHC class I Ags. The lack of induced
MHC class II expression on the ES cell-derived HPCs after IFN-␥
stimulation likely reflects the inability of IFN-␥ to up-regulate the
expression of CIITA, the master regulator for the transcription of
MHC class II genes, in ES-derived cells as reported by Ladhoff et
al. (33).
FIGURE 4. NK cell depletion augments HPC engraftment. A, Percentage of NK cells in the peripheral blood of Rag2⫺/⫺ pre and post i.p. administration of anti-NK1.1 mAb or isotype control mAb on days ⫺3 and
⫺1 before HPC transplantation. NK cells were detected using the DX5
mAb specific for the pan-NK marker CD49b. Data are represented as
mean ⫾ SEM; ⴱ, p ⬍ 0.05. B, HPCs were transplanted into anti-NK1.1 or
isotype control-treated Rag2⫺/⫺ mice and the percentage of HPC derived
cells in peripheral blood was monitored by flow cytometry for GFP expression on days 7, 14, and 28. Representative flow plots from individual
mice are shown. C, Summary of the percentage of HPC-derived cells in the
peripheral blood of anti-NK1.1 (n ⫽ 3) or isotype control-treated (n ⫽ 3)
Rag2⫺/⫺ recipient mice from day 7 to 70 post transplant. Data are represented as mean ⫾ SEM; ⴱ, p ⬍ 0.05.
To determine whether MHC class I expression protected HPCs
from NK cell-mediated killing in vivo, HPCs treated with IFN-␥
for 48 h were coinjected with nontreated HPCs at a 1:1 ratio into
Rag2⫺/⫺␥c⫺/⫺ and Rag2⫺/⫺ mice. To discriminate between the
two HPC populations by flow cytometry, the IFN-␥ treated HPCs
were prelabeled with the membrane dye PKH26, while the nontreated HPCs remained unlabeled. Splenocytes were harvested at
1, 6, and 24 h post transplantation, and the GFP⫹ population was
gated and further analyzed for PKH26. These early timepoints
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Rag2⫺/⫺ mice that had been treated with poly I:C and additionally
transplanted with HPCs 24 h before harvest. Indeed, the amount of
detectable granzyme B in these animals was less than that observed in splenocytes harvested from animals treated with poly I:C
alone (Fig. 3B), confirming that NK cells degranulate in the presence of target cells with the subsequent loss of detectable granzyme B by Western blotting.
To further investigate the activation status of NK cells following
HPC transplant, we harvested splenic NK cells from Rag2⫺/⫺
mice 6 h post HPC transplantation. The NK cells were then cocultured with HPCs overnight and the production of IFN-␥ by the NK
cells was measured by ELISPOT. The amount of IFN-␥ produced
by NK cells harvested from HPC-transplanted Rag2⫺/⫺ mice was
significantly higher than that produced by NK cells harvested from
naive animals (Fig. 3C), further confirming that HPC transplantation functionally activates NK cells. In addition, we assessed the
phenotype of NK cells 24 h post HPC-transplantation; however, no
differences were observed in the expression of DX5, NKG2D,
B220, CD16, CD25, CD44, CD62L, or CD69 when compared with
that of naive NK cells (supplementary Fig. 2). The lack of any
changes in the phenotype of NK cells could reflect possible differences in the kinetics of cytokine production and cell surface
expression of phenotypic markers.
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NK CELLS TARGET ES CELL-DERIVED HEMATOPOIETIC CELLS
plant, indicating that while the NK cells in Rag2⫺/⫺ recipient mice
could delete HPCs in vivo, they were incapable of killing the HPC
subpopulation that expressed MHC class I. The percentage of nontreated HPCs was further reduced at 6 h, and by 24 h the majority
of nontreated HPCs had been eliminated by NK cells. Analysis of
the bone marrow compartment 24 h post transplant demonstrated
that only HPCs pretreated with IFN-␥ could be detected in
Rag2⫺/⫺ recipients (Fig. 5C), consistent with the results observed
in the spleen (Fig. 5B). Our findings confirm that a large population of ES cell-derived HPCs is susceptible to NK cell-mediated
killing. Furthermore, these results suggest that while lack of MHC
expression by ES-derived cells may be advantageous in preventing
immunological sensitization, lack of MHC expression may not
protect against NK cell recognition.
HPC-derived Lin⫺ c-kit⫹ and Lin⫺ Sca-1⫹ progenitors are
early targets of NK cell killing
were chosen for analysis because NK killing of transplanted bone
marrow cells has been shown to occur within 24 h (26). In the
Rag2⫺/⫺␥c⫺/⫺ mice both the IFN-␥-treated HPCs and the nontreated HPCs were detected at the original input ratio of 1:1 at all
time points tested (Fig. 5B). In stark contrast, an elevated percentage of IFN-␥-treated HPCs and a reduced percentage of nontreated
HPCs were detected in Rag2⫺/⫺ mice as early as 1 h post trans-
Role of H60 in regulating long-term engraftment of HPCs
Next, we analyzed the c-kit⫹ and Sca-1⫹ HPCs by flow cytometry
for the expression of NK cell ligands. Indeed, the majority of the
c-kit⫹ and Sca-1⫹ HPCs expressed H60, while the expression of
H-2Kb was virtually absent (Fig. 6B). Furthermore, we measured
NKG2D ligand expression on HPCs recovered from Rag2⫺/⫺␥c⫺/⫺
recipients 24 h post transplant because it was unlikely that the
observed reduction in HPC engraftment in Rag2⫺/⫺ recipients
compared with Rag2⫺/⫺␥c⫺/⫺ recipients was due solely to the
deletion of c-kit⫹ and Sca-1⫹ HPCs. Consistent with this notion,
we observed an up-regulation of Rae-1 on HPCs in vivo while H60
expression was maintained (Fig. 6C). These findings suggest that
following transplantation, HPCs up-regulate Rae-1 in addition to
the already high levels of H60, thus increasing their susceptibility
to NK cell-mediated killing. This Rae-1 up-regulation could be
induced by serum cytokines or stress factors post transplant.
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FIGURE 5. MHC class I expression protects HPCs from NK cell cytotoxicity in vivo. A, Percentage of HPCs expressing the MHC class I molecule H-2Kb (䡺) or the MHC class II molecules IA/IE (f) after in vitro
culture with 20 ng/ml IFN-␥ for 0, 24, 48, or 72 h. One representative
experiment of three is shown. B, An in vivo NK cytotoxicity assay was
performed by injecting Rag2⫺/⫺␥c⫺/⫺ and Rag2⫺/⫺ mice with target cells
consisting of 107 GFP⫹ HPCs and 107 GFP⫹PKH26-labeled HPCs that
had been treated with IFN-␥ for 48 h. Representative flow plots of the
GFP⫹PKH26-unlabeled and GFP⫹PKH26-labeled cells recovered from
the spleens of Rag2⫺/⫺␥c⫺/⫺ or Rag2⫺/⫺ mice at 1, 6, and 24 h after
injection are shown. The percentages of GFP⫹PKH26-unlabeled and
GFP⫹PKH26-labeled cells are indicated. Results from one representative
experiment of two are shown. C, Representative flow plots of the
GFP⫹PKH26-unlabeled and GFP⫹PKH26-labeled cells recovered from
the bone marrow of Rag2⫺/⫺␥c⫺/⫺ or Rag2⫺/⫺ mice 24 h after injection
are shown. The percentages of GFP⫹PKH26-unlabeled and GFP⫹PKH26labeled cells are indicated. Results from one representative experiment of
two are shown.
Our data thus far indicated that while a subpopulation of HPCs is
capable of engrafting in Rag2⫺/⫺ recipient mice, that subpopulation fails to maintain life-long hematopoiesis. One possible explanation for the observed decline of HPC-derived cells in Rag2⫺/⫺
recipients is that NK cells continue to eliminate HPCs at late time
points post transplant; however, this scenario is unlikely because it
has been demonstrated that NK cells only play a role in the rejection of allogeneic bone marrow within the first week post transplant (26, 31). The simplest alternate explanation is that the subpopulation of HPCs deleted by NK cells early post transplant
contains the long-term hematopoietic progenitors capable of maintaining permanent engraftment, while the engrafting population is
comprised of mature progenitors that have limited self-renewal
capacity. To examine this further, we transplanted HPCs into
Rag2⫺/⫺␥c⫺/⫺ recipient mice and analyzed bone marrow cells after 24 h to determine whether HPCs expressing c-kit and Sca-1,
markers of hematopoietic stem cells, migrate to and populate hematopoietic stem cell niches. The 24-h timepoint was chosen because the majority of NK cell mediated killing had occurred by
24 h as shown in Fig. 5B above. Interestingly, the majority of
HPC-derived cells detected in the bone marrow expressed c-kit,
and a smaller number expressed Sca-1, suggesting that the c-kit
and Sca-1 expressing HPCs were the earliest migrants to the bone
marrow niches (Fig. 6A). Furthermore, the HPC-derived c-kit⫹ and
Sca-1⫹ cells detected within the bone marrow of Rag2⫺/⫺␥c⫺/⫺
recipients 24 h posttransplant did not express the lineage markers
CD3, B220, CD11c, Mac-1, or Gr-1, suggesting that the HPCderived c-kit⫹ and Sca-1⫹ cells detected in the bone marrow were
phenotypically similar to long-term hematopoietic stem cells.
The Journal of Immunology
5455
To directly examine whether Lin⫺c-kit⫹ and Lin⫺Sca-1⫹ HPCs
could be deleted by NK cells, we transplanted HPCs into Rag2⫺/⫺
or Rag2⫺/⫺␥c⫺/⫺ recipient mice and analyzed their bone marrow
cells for the presence of HPC-derived c-kit⫹ and Sca-1⫹ cells after
24 h. A total of 2 ⫻ 106 bone marrow events per animal (n ⫽ 3
animals per group) were acquired by flow cytometry, and GFP⫹
HPC-derived cells were gated and further analyzed for c-kit and
Sca-1 expression. In Rag2⫺/⫺␥c⫺/⫺ recipients, GFP⫹c-kit⫹ and
GFP⫹Sca-1⫹ cells were readily detected and comprised ⬃0.02%
of the total bone marrow cells (Fig. 6D). In stark contrast, the
number of GFP⫹c-kit⫹ and GFP⫹Sca-1⫹ cells detected in
Rag2⫺/⫺ recipients was significantly reduced. Taken together,
these data indicate that the c-kit⫹ and Sca-1⫹ HPC subpopulations
are able to home to the bone marrow and may contribute to longterm hematopoietic engraftment in an environment devoid of NK
cells; however, in the presence of NK cells, the c-kit⫹ and Sca-1⫹
cells are rapidly deleted and fail to engraft in the bone marrow
niches.
Finally, to determine the impact of H60 on HPC killing by NK
cells, we blocked H60 on HPCs with a mAb and then measured
NK cell killing of H60-blocked HPCs with a flow cytometric cytotoxicity assay. H60 blocking significantly inhibited the ability of
NK cells to kill HPCs in a dose-dependent manner (Fig. 6E); however, complete inhibition was not observed, even at the highest Ab
concentration tested. This suggested that additional NK stimulatory ligands may be involved. Interestingly, blockade of Rae-1
alone, or in combination with H60 did not further inhibit NK cell
killing of HPCs (data not shown), suggesting that NK cells could
also kill HPCs via an NKG2D-independent mechanism. Overall,
our data indicate for the first time that NK cells form a significant
barrier to the engraftment of ES cell-derived cells.
Discussion
The ability of ES cells to differentiate into functional cells and
tissues of all three germ layers has raised hope that ES cells can be
used to treat degenerative diseases. Unfortunately, immunological
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FIGURE 6. HPC-derived Lin⫺c-kit⫹ and Lin⫺Sca-1⫹ progenitors are target cells for NK cells. A, Flow cytometric analysis of HPC-derived c-kit⫹ and
Sca-1⫹ cells found in the bone marrow post HPC-transplantation for the expression of lineage markers. 107 HPCs were transplanted into Rag2⫺/⫺␥c⫺/⫺
mice. Twenty-four hours post transplant, HPC-derived cells within the bone marrow were distinguished from the host’s endogenous bone marrow cells on
the basis of GFP expression. The GFP⫹ and GFP⫺ bone marrow cells were independently gated and further analyzed for expression of the lineage markers
CD3, B220, CD11c, Mac-1, and Gr-1. Results from one representative experiment of two are shown. B, Flow cytometric analysis of HPC-derived c-kit⫹
and Sca-1⫹ cells post in vitro differentiation for the expression of H-2Kb and H60. c-kit⫹ and Sca-1⫹ cells express H60. Results from one representative
experiment of three are shown. C, Comparison of HPC expression of NKG2D ligands in vitro and in vivo. 107 HPCs were transplanted into Rag2⫺/⫺␥c⫺/⫺
mice (n ⫽ 3). Twenty-four hours post transplant, HPC-derived cells within the bone marrow were distinguished from the host’s endogenous bone marrow
cells on the basis of GFP expression and further analyzed for MULT-1, Rae-1, and H60 expression. Data are represented as mean ⫾ SEM. D, 107 HPCs
were transplanted into Rag2⫺/⫺␥c⫺/⫺ or Rag2⫺/⫺ mice. Twenty-four hours post transplantation, bone marrow GFP⫹ cells were gated and further analyzed
for expression of c-kit and Sca-1. A summary of the total c-kit⫹ and Sca-1⫹ cell counts per 2 ⫻ 106 bone marrow cells acquired is shown (n ⫽ 3 animals
per group). Both cell types are deleted in Rag2⫺/⫺ but not in Rag2⫺/⫺␥c⫺/⫺ mice. Data are represented as mean ⫾ SEM; ⴱ, p ⬍ 0.05. E, Blocking H60
inhibits NK cell-mediated killing of HPCs. A flow based cytotoxicity assay was performed using splenocytes derived from polyI:C treated Rag2⫺/⫺ mice
as the source of NK effectors against HPC target cells at an E:T ratio of 20 to 1. Anti-H60 blocking Ab or isotype control was added at the indicated
concentrations. Percent inhibition corresponds to the reduction in target cell lysis compared with controls with no Ab. The mean inhibition of duplicate
assays from one representative experiment of two is shown.
5456
NK CELLS TARGET ES CELL-DERIVED HEMATOPOIETIC CELLS
required to maintain the permanent engraftment of ES
cell-derived HPCs.
Alternatively, treating HPCs with IFN-␥ led to MHC class I
up-regulation, which protected HPCs from NK cell-mediated cytotoxicity. However, the caveats of this strategy include the unknown effects of IFN-␥ on progenitor cell self-renewal properties
and the obvious potential triggering of T- and B cell alloresponses
in immunocompetent mice. Under current conditions, ⬃15–25%
of HPCs express appreciable levels of MHC class I after in vitro
differentiation. These MHC class I positive cells likely comprise
the subpopulation of HPCs capable of transient engraftment in
Rag2⫺/⫺ recipients. As ES cells differentiate, they gain expression
of MHC class I and lose their plasticity and ability to self-renew
(10). Thus, it remains plausible that HPCs expressing MHC class
I no longer possess the self-renewal capabilities of true stem cells.
Bhattacharya et al. (35) reported ⬃0.1–1.0% of the rare hematopoietic stem cell niches are available for engraftment at any
given time in nonconditioned recipient mice. Consistent with these
findings, 0.02% of the cells detected in the bone marrow of nonconditioned Rag2⫺/⫺␥c⫺/⫺ recipients 24 h post transplant were
HPC-derived. Importantly, the HPC-derived cells detected in the
bone marrow did not express lineage markers, but they did express
either c-kit or Sca-1, phenotypic characteristics of long-term hematopoietic stem cells. These data suggest that HPC-derived
Lin⫺c-kit⫹ and Lin⫺Sca-1⫹ cells engraft in the available hematopoietic stem cell niches with similar efficiency as true hematopoietic stem cells and may be sufficient to sustain life-long hematopoiesis. Although Lin⫺c-kit⫹ and Lin⫺Sca-1⫹ HPCs were
readily detected, we did not observe any Lin⫺c-kit⫹Sca-1⫹ ES
cell-derived HPCs (data not shown). The lack of Lin⫺ckit⫹Sca-1⫹ HPCs suggests that the in vitro method used to differentiate ES cells into hematopoietic precursors does not fully replicate in utero development. However, HPC transplantation
rescues long-term hematopoiesis in lethally irradiated adult animals (7, 8, 36); therefore, it remains possible that after transplantation, the in vivo environment further instructs Lin⫺c-kit⫹ and
Lin⫺Sca-1⫹ HPCs to fully develop into Lin⫺c-kit⫹Sca-1⫹ cells.
Our data also demonstrate that HPC-derived Lin⫺c-kit⫹ and
Lin⫺Sca-1⫹ cells are excellent NK cell targets because robust populations of HPC-derived c-kit⫹ and Sca-1⫹ cells were detected in
the bone marrow of NK-deficient Rag2⫺/⫺␥c⫺/⫺ mice, but not in
NK-replete Rag2⫺/⫺ recipients. It has recently been demonstrated
that NK cells directly reject MHC class I deficient hematopoietic
stem cells (23). Consistent with this finding, Lin⫺c-kit⫹ and
Lin⫺Sca-1⫹ HPCs do not express MHC class I and are thus likely
to behave similarly to class I deficient hematopoietic stem cells.
NK cells reject allogeneic bone marrow cells, yet their contribution to the rejection of solid organ allografts may be limited (37);
however, in a recent study using the Rag⫺/⫺ and Rag2⫺/⫺␥c⫺/⫺
mouse models, Kroemer et al. (38) reported that NK cells activated
in vivo by IL-15 could mediate the acute rejection of skin allografts in the absence of any adaptive immune cells. The ability of
NK cells to reject ES cell-derived HPCs may reflect the access NK
cells have to the lymphoid organs where HPCs migrate. Whether
NK cells can also mediate the rejection of ES cell-derived cells or
tissues other than hematopoietic cells remains to be determined;
however, preliminary data from our laboratory show that ES cellderived cardiomyocytes and definitive endodermal cells express
low levels of MHC class I Ags (data not shown), suggesting that
they too may be targets of NK cells after transplantation. Furthermore, Wu et al. (39) recently examined the immune response
against ES cell-derived insulin-producing cells in immunocompetent allogeneic recipients. Although graft function ultimately
failed, it also unexpectedly failed in immunocompetent syngeneic
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studies of ES cell derivatives have been largely ignored, yet they
are imperative before ES derivatives can be applied clinically. In
this study, we used the unique characteristics of Rag2⫺/⫺ (B and
T cell deficient) and Rag2⫺/⫺␥c⫺/⫺ (B, T, and NK cell deficient)
mice to assess the ability of NK cells to regulate ES-derived HPC
engraftment. We demonstrate that NK cells rapidly reject HPCs
within the first 24 h post transplant, comparable to the rejection
kinetics observed in allogeneic bone marrow transplantation (26).
Although a subpopulation of HPCs survives the NK cell response,
engraftment is transient, suggesting lack of self renewal properties.
Importantly, our results show that engraftment failure correlates
with a reduction of HPC-derived c-kit⫹ and Sca-1⫹ cells in the
bone marrow niche of NK-rich Rag2⫺/⫺ mice. Although we did
not exhaustively study myeloid and lymphoid engraftment, we can
infer from our data that NK cells equally regulate both myeloid
and lymphoid progenitor engraftment because the engraftment of
all HPC-derived cells ultimately fails. Further, our previous data
indicate that the majority of HPCs generated in this model form
myeloid rather than lymphoid cells, a characteristic attributed to
HOXB4.
Several studies have used ES cell expression of MHC class I and
class II combined with in vitro assays, such as the MLR or 51Cr
release assay, as measures of ES cell immunogenicity (10, 11, 13,
32). In accordance with these reports, ES cell-derived HPCs expressed low levels of MHC class I and virtually no MHC class II
Ags. HPCs also expressed high levels of H60, an activating NK
cell ligand, suggesting a potential mechanism for NK cell recognition and lysis of HPCs. Using 51Cr release assays, previous studies, including ours, demonstrated that undifferentiated ES cells and
embryonic-derived neural stem cells are not susceptible to NK
cell-mediated killing in vitro (10, 13, 34). These studies concluded,
on the basis of lack of MHC expression and negative in vitro cell
lysis data, that ES cells and their derivatives are immune privileged. In contrast, we demonstrate that purified CD45⫹ ES cellderived HPCs are susceptible to NK cell cytotoxicity. This discrepancy may be due to the greater sensitivity of the flow-based
assay compared with the 51Cr release assay (27) and due to the fact
that these current studies are the first to analyze purified cells
rather than undifferentiated ES cells or EB-derived cells. Importantly, disrupting the interaction of H60 on HPCs with NKG2D on
NK cells with a blocking anti-H60 Ab inhibited the ability of NK
cells to lyse HPCs, highlighting the role of H60 expression in
enabling NK cells to regulate HPC engraftment in vivo. Furthermore, we confirmed that lack of MHC class I expression makes
ES-derived cells excellent targets of NK cell killing in vivo. We
conclude that results of low MHC class I and class II expression
and a lack of killing observed with 51Cr release assays in vitro are
inadequate parameters for determining the fate of ES cell-derived
cells in vivo.
The data presented in this study further underline the notion that
NK cells are sufficient to mediate rejection of transplanted ES cellderived HPCs, without T or B cell involvement. Consequently,
transplantation of ES cell-derived HPCs triggers the activation of
resting NK cells, as evidenced by the up-regulation of perforin and
granzyme B in NK cells. Tian et al. (32) recently demonstrated that
depletion of NK cells using an anti-ASGM1 Ab in NOD/SCID
mice improved engraftment of human ES cell-derived hematopoietic cells in a xenotransplantation model. In this study, a preconditioning regimen using anti-NK1.1 mAb to deplete NK cells in
addition to irradiation before transplant significantly augmented
HPC engraftment, with peak levels of engraftment reaching those
observed in NK cell-deficient mice; however, this improved engraftment was not permanently sustained. These results suggest
that continued administration of an NK cell-depleting Ab may be
The Journal of Immunology
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Disclosures
The authors have no financial conflict of interest.
23.
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recipients, suggesting a potential role for innate immune mechanisms in contributing to graft failure (39). However, the role of NK
cells in regulating the engraftment of ES cell-derived insulin-producing cells in this model system remains to be determined.
In summary, our data challenge the belief that ES cells and their
derivatives are immune privileged by virtue of low MHC Ag expression (10 –13). Although advantageous to avoiding T and B cell
responses, poor MHC class I expression in combination with high
level expression of H60 is detrimental to ES cell-derived HPC
engraftment due to NK cell recognition and killing. Our findings
provide direct evidence that NK cells, a previously overlooked
lymphocyte subset, are a significant immunological barrier to the
in vivo engraftment of ES cell-derived HPCs. However, these
studies were performed in Rag2⫺/⫺ mice, with ⬎50 – 60% of
splenocytes being NK cells, which is much higher than the 1–3%
detected in immunocompetent mice. Thus, the impact of NK cells
in immunocompetent mice may be less severe. These studies are
ongoing. Despite that caveat, transplantation of ES cell-derived
progenitor cells may require the control of NK cells to be
successful.
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