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IMMUNOBIOLOGY
Inefficient response of T lymphocytes to glycosylphosphatidylinositol
anchor–negative cells: implications for paroxysmal nocturnal hemoglobinuria
Yoshiko Murakami, Hiroshi Kosaka, Yusuke Maeda, Jun-ichi Nishimura, Norimitsu Inoue, Kazuhito Ohishi,
Masaru Okabe, Junji Takeda, and Taroh Kinoshita
Paroxysmal nocturnal hemoglobinuria
(PNH) is a hematopoietic stem cell disorder in which clonal cells defective in
glycosylphosphatidylinositol (GPI) biosynthesis are expanded, leading to
complement-mediated hemolysis. PNH is
often associated with bone marrow suppressive conditions, such as aplastic anemia. One hypothetical mechanism for the
clonal expansion of GPIⴚ cells in PNH is
that the mutant cells escape attack by
autoreactive cytotoxic cells that are
thought to be responsible for aplastic
anemia. Here we studied 2 model systems. First, we made pairs of GPIⴙ and
GPIⴚ EL4 cells that expressed major histocompatibility complex (MHC) class II
molecules and various types of ovalbumin. When the GPI-anchored form of
ovalbumin was expressed on GPIⴙ and
GPIⴚ cells, only the GPIⴙ cells presented
ovalbumin to ovalbumin-specific CD4ⴙ T
cells, indicating that if a putative autoantigen recognized by cytotoxic cells is a
GPI-anchored protein, GPIⴚ cells are less
sensitive to cytotoxic cells. Second, antigen-specific as well as alloreactive CD4ⴙ
T cells responded less efficiently to GPIⴚ
than GPIⴙ cells in proliferation assays. In
vivo, when GPIⴚ and GPIⴙ fetal liver cells,
and CD4ⴙ T cells alloreactive to them,
were cotransplanted into irradiated hosts,
the contribution of GPIⴚ cells in peripheral blood cells was significantly higher
than that of GPIⴙ cells. The results obtained with the second model suggest
that certain GPI-anchored protein on target cells is important for recognition by T
cells. These results provide the first experimental evidence for the hypothesis
that GPIⴚ cells escape from immunologic
attack. (Blood. 2002;100:4116-4122)
© 2002 by The American Society of Hematology
Introduction
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by the presence of clonal
blood cells deficient in glycosylphosphatidylinositol (GPI)–
anchored proteins. A somatic mutation of the PIGA gene, which is
involved early in the biosynthesis of the GPI anchor,1 is responsible
for the deficiency.1-3 Because GPI acts as a membrane anchor for
many cell surface proteins,4 a loss of function of PIGA results in the
loss of all the GPI-anchored proteins from the blood cell surface.
PIGA is X-linked; therefore, one hit of somatic mutation in PIGA
causes GPI anchor deficiency in the hematopoietic stem cell.2
Clonal cells derived from the mutant hematopoietic stem cell
occupy a big fraction of hematopoietic cells.5
The characteristic clinical triad of PNH includes hemolysis,
venous thrombosis, and bone marrow failure.6,7 The activation of
complement leads to the lysis of red blood cells deficient in the
GPI-anchored proteins CD59 and CD55 that protect host cells from
the attack by complement.4,5 An impaired regulation of complement activation may also be relevant to venous thrombosis.6,7 Bone
marrow failure may be due to an autoimmune mechanism as
thought to operate in idiopathic aplastic anemia (AA) that is often
associated with PNH.6,7
Clonal expansion of cells derived from the mutant hematopoietic
stem cell is critical for clinical manifestation of PNH. The mechanism,
however, has not been elucidated. To test whether a defective PIGA
alone can cause the clonal expansion, we as well as Rosti and colleagues
disrupted the mouse Piga gene, a homologue of PIGA, in embryonic
stem cells and raised chimeric mice bearing GPI⫺ cells.8,9 Because
GPI-anchored proteins are essential for development,10 mice with high
chimerism did not survive. The analysis of a limited number of mice
with very low levels of chimerism hardly showed the autonomous
increase of GPI⫺ cells. The chimeric mice, however, had GPI⫺ cells in
nonhematopoietic tissues, whereas patients with PNH have GPI⫺ cells
only in the hematopoietic system.
To overcome these problems, we made PNH model mice bearing
GPI⫺ cells only in the hematopoietic system by a combination of
Cre/loxP-mediated disruption of Piga in embryos and transplantation of
their fetal liver cells into irradiated hosts.Analysis of these mice revealed
that the percentage of GPI⫺ cells remained constant; the Piga mutation
alone does not account for the clonal expansion of the mutant stem cell
and the second factor must be involved in the pathogenesis of PNH.11
The same conclusion was obtained from the different Cre/loxPmediated Piga-disrupted mice.12 The report that most healthy individuals have a small number of PIGA mutant granulocytes is consistent with
this conclusion.13
Then, there have been 2 hypotheses about the second factor.
One holds that the mutant clone itself gains an intrinsic ability to
From the Department of Immunoregulation, Research Institute for Microbial
Diseases, Department of Experimental Genome Research, Genome
Information Research Center, and Departments of Dermatology and
Environmental Medicine, Graduate School of Medicine, Osaka University,
Suita, Osaka, Japan.
and Technology of Japan.
Reprints: Taroh Kinoshita, Department of Immunoregulation, Research
Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita,
Osaka 565-0871, Japan; e-mail: [email protected].
Submitted June 6, 2002; accepted July 4, 2002. Prepublished online as Blood
First Edition Paper, July 18, 2002; DOI 10.1182/blood-2002-06-1669.
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.
Supported by grants from the Ministry of Education, Culture, Sports, Science
© 2002 by The American Society of Hematology
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BLOOD, 1 DECEMBER 2002 䡠 VOLUME 100, NUMBER 12
expand, a growth phenotype, by one or more additional genetic
modifications. The other holds that the mutant clone is selected
under the affected environment.6 As to the latter hypothesis, it was
suggested that the affected environment may be an autoimmune
process,14 because this is a widely accepted pathogenetic mechanism in idiopathic AA.15 The effector responsible in AA has not
been clarified, but there are reports that CD4⫹ T-cell clones capable
of killing autologous hematopoietic progenitor cells can be generated by culturing T cells of AA patients with autologous hematopoietic progenitor cells.16,17 PNH, like AA, is reported to be strongly
associated with the HLA haplotype DR218,19 and skewed usage of
T-cell receptor (TCR) V␤ genes.20,21 So, we assumed CD4⫹ T cells
to be cytotoxic effectors that inhibit the hematopoiesis of normal
stem cells. If this is true, there must be some difference in
sensitivity to cytotoxic T cells (CTLs) between GPI⫹ and GPI⫺
cells. Here, we test this hypothesis experimentally and, for the first
time, show that the GPI⫺ cell population could expand escaping
CTL attack in an in vivo system.
Materials and methods
Mice
Mice transgenic for ovalbumin (OVA)–specific TCR (termed OTII)22 were
donated by Dr F. R. Carbone and W. R. Heath (Monash Medical School, and
the Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). The Ly5.1 congeneic line of C57BL/6 (B6) mice was kindly provided
by Dr H. Nakauchi (Tsukuba University, Ibaragi, Japan). B6 and B6.CH2bm12/KhEg (bm12) mice were obtained from Japan SLC (Shizuoka,
Japan) and the Jackson Laboratory (Bar Harbor, ME), respectively. The
experiments with mice included in this study were approved by the Review
Board of the Research Institute for Microbial Diseases, Osaka University.
Peptides and antibodies
Peptide ISQAVHAAHAEINEAGR (OVA323-339) was purchased from Peptide Institute (Osaka, Japan). Fluorescein isothiocyanate (FITC)–conjugated anti-Ly5.1, phycoerythrin (PE)–conjugated anti–heat-stable antigen
(HSA), biotinylated anti–I-Ab␤, -HSA, and -DX-5, allophycocyaninconjugated anti-CD8 and streptavidin, and peridinin chlorophyll protein
(PerCP)–streptavidin were purchased from Pharmingen (San Diego, CA).
Anti-B220 (RA3-6B2), anti–Mac-1 (M1/70), anti-CD4 (GK1.5), anti-CD8
53-6.7, and anti-TER119 were purified from each antibody-producing
hybridoma (from Dr T. Kina, Kyoto University, Kyoto, Japan) and
biotinylated.
Cell lines
A vector for the expression of GPI-anchored OVA (GPI-OVA) was
constructed by 4 parts ligation of an EcoRI-PstI fragment encoding the
N-terminal signal sequence of human CD59, a polymerase chain reaction
(PCR)–amplified fragment corresponding to nucleotides 415-1161 of
chicken OVA cDNA (to which PstI and XhoI sites were attached), an
XhoI-NotI fragment encoding the human CD59 GPI attachment signal, and
EcoRI- and NotI-cut pME18sf-neo, a mammalian expression plasmid. A
vector for the expression of transmembrane OVA (TM-OVA) was constructed connecting EcoRI- and XbaI-cut pME18sf-neo, the same first 2
fragments used to generate the GPI-OVA vector, and a PCR-amplified
fragment encoding a transmembrane region of human membrane cofactor
protein (MCP; nucleotides 953-1203 of MCP cDNA of STBC/CYT2
allotype, a gift from Dr T. Seya, Osaka Medical Center for Cancer and
Cardiovascular Diseases, Osaka, Japan), to which XhoI and XbaI sites are
attached. A vector pAc-Neo-OVA bearing native OVA cDNA was a gift
from Dr T. Sato (Tokai University, Kanagawa, Japan). Expression vectors
for I-Ab ␣ and ␤ chains were kindly provided by Dr L. Karlsson (R. W.
Johnson Pharmaceutical Research Institute, San Diego, CA). A vector that
expresses mouse Pigf cDNA was reported previously.23
RESPONSE OF T CELLS TO GPI ANCHOR–NEGATIVE CELLS
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We first stably transfected a Pigf-deficient clone of mouse thymoma cell
line (EL4 Pigf⫺)24 with vectors for I-Ab ␣ and ␤ chains and chose one clone
with a high surface expression of I-Ab. We then stably transfected that clone
with vectors expressing various types of OVA and selected one clone each.
We further transfected the selected clones stably with either a Pigfexpressing or a control vector to obtain pairs of GPI⫹ and GPI⫺
transfectants expressing various forms of OVA. To generate cells used in
OVA-peptide pulse and allogeneic response experiments, the transfection
with OVA-expression vector was omitted. To avoid clonal variation, the
GPI⫹ and GPI⫺ pairs were used as bulk populations. All the transfectants
were cultured in Dulbecco modified Eagle medium (DMEM; Gibco,
Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS) and 100
U/mL penicillin and 100 ␮g/mL streptomycin.
Cell purification
Highly purified CD4⫹ T cells (⬎ 95% pure as assessed by fluorescenceactivated cell sorting [FACS]) were prepared from pooled lymph nodes of
OTII or bm12 mice by hypotonically lysing erythrocytes, followed by
negative selection using the MACS system (cells treated with a cocktail of
biotinylated anti-CD8, -HSA, -I-Ab␤, and -B220 monoclonal antibodies
[mAbs], followed by streptavidin beads; Miltenyi Biotec, Bergish Gladbach, Germany). Bone marrow cells, flushed from femurs, were depleted of
lineage-marker positive cells by negative selection using the MACS system
(cells treated with a cocktail of anti-CD4, -CD8, -B220, -Mac-1, and -DX-5
mAbs, followed by streptavidin beads).
In vitro T-cell proliferation
Purified CD4⫹ T cells from OTII or bm12 mice (3 ⫻ 105/well) were mixed
with various types of EL4 transfectants (5 ⫻ 105/well) in flat-bottom
96-well plates in DMEM supplemented with 10% FCS, 2 mM L-glutamine,
5 ⫻ 10⫺5 M 2-mercaptoethanol, and 100 U/mL penicillin and 100 ␮g/mL
streptomycin. The EL4 cells were pretreated with 50 ␮g/mL mitomycin C
(Sigma Chemical, St Louis, MO) for 1 hour and washed 3 times with the
medium. For some experiments, they were pulsed with OVA peptides (5 or
10 ␮M). Proliferation was assessed on days 4 and 5 by pulsing cultures with
[3H]thymidine for 4 hours.
In vivo experimental model of PNH
Mice transgenic for hCMV-Cre were generated in the B6 strain according to
a previous report.25 Mice with a Piga gene bearing loxP sites within intron 5
and the 3⬘ flanking region (termed Pigaflox) were reported previously.26 The
generation of Piga-disrupted hematopoietic stem cells using these 2 mouse
strains was also reported.11 Briefly, we crossed Pigaflox males with the
hCMV-Cre transgenic females. All female embryos should be heterozygous
for Pigaflox and among them those that received the Cre transgene would
become heterozygous for Piga disruption and mosaic for Pig-a expression
due to random X inactivation. We collected fetal liver cells on 14 days after
coitus (dac) as a source of stem cells and transferred them (3 ⫻ 106/mouse)
intravenously into lethally irradiated B6 hosts (900 cGy irradiation from a
137Cs source). We cotransplanted them with or without purified CD4⫹ T
cells of bm12 mice (7.5 ⫻ 104 cells/mouse) together with lineage marker–
negative bm12 bone marrow cells (106 cells/mouse) to prevent death from
aplasia.27 To distinguish fetal liver–derived cells from other cells, we used
mice whose Ly5 allele was 5.1 for the donor of fetal liver and 5.2 for the
host and bm12 mice. Flow cytometric analysis of the peripheral blood cells
of these chimeric mice was reported previously.11
Results
We tested 2 models to obtain experimental evidence for the
hypothesis that expansion of the GPI⫺ cell population seen in PNH
is caused by survival of GPI⫺ cells in the presence of autoreactive T
cells.6,14 We focused on CD4⫹ T cells.28 In the first model, we tested
a specialized situation where a putative autoantigen recognized by
CD4⫹ T cells is derived from GPI-anchored proteins (Figure 1A).
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When GPI anchors are not attached to proteins that are normally
GPI anchored, the proteins are not expressed on the cell surface due
either to degradation in the cytoplasm or to secretion into the
extracellular space.4 Peptides generated in the cytoplasm would not
be presented on MHC class II molecules. To test whether OVA
destined for extracellular secretion is presented on MHC class II
molecules, we used GPI⫹ and GPI⫺ EL4 cells expressing nativesecreted OVA (native OVA in Figure 2). The antigenic peptide was
not presented on class II molecules irrespective of the presence of
GPI (shaded and black bars). Taken together with the above result
that GPI⫺ cells presented OVA peptide from TM-OVA, it is
suggested that the cell surface expression of GPI-anchored proteins
is required for the presentation of antigenic peptides on MHC class II
molecules. These results indicate that if a putative autoantigen recognized by CD4⫹ T cells is derived from GPI-anchored proteins, GPI
anchor–deficient cells would be more insensitive to CD4⫹ T-cell–
mediated cytotoxicity than GPI anchor–sufficient cells.
GPI-anchored protein-negative cells are less efficient than
GPI-anchored protein-positive cells in MHC class II–mediated
stimulation of CD4ⴙ T cells
Figure 1. Two experimentally tested models. (A) A putative autoantigen recognized
by CD4⫹ T cells is derived from GPI-anchored proteins. GPI⫹ cells (left) process
GPI-anchored proteins and present antigenic peptides on MHC class II molecules,
whereas GPI⫺ cells (right) do not present such antigenic peptides. (B) Some GPI-anchored
protein is a ligand for some costimulatory molecules on CD4⫹ T cells. GPI⫹ cells (left)
efficiently stimulate CD4⫹ T cells, whereas GPI⫺ cells (right) do not.
In the second model, we tested whether a lack of GPI-anchored
proteins on antigen-presenting cells (APCs; or target cells) affects
stimulation of (or recognition by) CD4⫹ T cells (Figure 1B).
Cells defective in GPI biosynthesis are unable to present
antigenic peptides of GPI-anchored proteins on MHC
class II molecules
To test whether antigenic peptides derived from endogenous GPIanchored proteins are presented on MHC class II molecules and if so
whether the presentation is dependent on GPI anchoring, we used the
GPI-anchored form of OVA as a model antigen. We isolated CD4⫹ T
cells from lymph nodes of OTII mice that express transgenic TCR
specific for an OVA-derived peptide presented on I-Ab molecules. These
T cells were stimulated with GPI⫹ and GPI⫺ EL4 cell lines expressing
I-Ab and various forms of OVA. We first used TM-OVA to confirm that
the GPI⫺ EL4 cell line is able to present peptides derived from OVA on
I-Ab molecules. The GPI⫺ EL4 cell line expressing TM-OVA (shaded
bar) stimulated the CD4⫹ T cells (Figure 2). The efficiency was about
one third that of the GPI⫹ EL4 cell line expressing TM-OVA (black bar;
34 000 versus 90 000 cpm; Figure 2). We then tested the presentation of
OVA peptides derived from GPI-OVA. The GPI⫹ EL4 cell line
expressing GPI-OVAstimulated the CD4⫹ T cells (black bar), indicating
that antigenic peptides derived from GPI-anchored proteins can be
presented on MHC class II molecules. In contrast, GPI⫺ EL4 transfected
with the same cDNA encoding GPI-OVA did not stimulate the CD4⫹ T
cells (shaded bar). The same GPI⫺ EL4 stimulated the CD4⫹ T cells
when exogenous OVA peptides were added (white bar), confirming that
GPI⫺ EL4 had an intact machinery for MHC class II–dependent antigen
presentation. Therefore, cells defective in GPI anchor biosynthesis are
not able to present antigenic peptides of GPI-anchored proteins on MHC
class II molecules.
As described above, the GPI⫺ EL4 cell line presented TM-OVA–
derived peptide significantly but less efficiently than the GPI⫹
counterpart (TM-OVA in Figure 2). FACS analysis showed that the
surface expression of TM-OVA and I-Ab on these 2 cell lines was
similar (mean fluorescent channels of GPI⫹ and GPI⫺ cells were
295 and 245 for TM-OVA, and 115 and 145 for I-Ab, respectively.).
Considering that a number of GPI-anchored proteins are involved
in cell-to-cell interactions, we next compared the stimulation of
CD4⫹ T cells in the presence and absence of GPI-anchored proteins
on APCs. To eliminate the intracellular antigen-processing step, we
compared GPI⫹ and GPI⫺ EL4 cell lines that express I-Ab
molecules but not TM-OVA. These cell lines were pulsed with
various concentrations of OVA peptides and then tested for their
ability to stimulate CD4⫹ T cells from OTII mice. Stimulation of
CD4⫹ T cells by GPI⫺ cells was significantly weaker than that by
the GPI⫹ counterpart (Figure 3). These results suggest that
GPI-anchored proteins costimulate antigen presentation by enhancing cell-to-cell interaction and that GPI⫺ cells would be less
sensitive to cytotoxic CD4⫹ T cells.
Figure 2. GPI anchor–negative cells do not present antigenic peptides of
GPI-anchored proteins on MHC class II molecules. CD4⫹ T cells isolated from a
TCR-transgenic mouse (OTII) were mixed with EL4 cells transfected with various forms of
OVA and treated with mitomycin C. Proliferation was assessed on day 4. TM-OVA indicates
EL4 cells transfected with the transmembrane form of OVA; GPI-OVA, EL4 cells
transfected with GPI-anchored OVA; native OVA, EL4 cells transfected with native OVA;
vector, vector-transfected EL4 cells; o, EL4 cells defective in GPI anchor biosynthesis;
f, EL4 cells with normal GPI anchor biosynthesis; and 䡺, GPI⫺ EL4 cells pulsed with OVA
peptide. Data (mean ⫹ SD of duplicate samples) were from a representative of 7
experiments, all with similar results.
BLOOD, 1 DECEMBER 2002 䡠 VOLUME 100, NUMBER 12
Figure 3. Some GPI-anchored proteins on APCs facilitate interaction with T
cells. Purified OTII-CD4⫹ T cells were incubated with I-Ab–expressing EL4 cells
pulsed with various concentrations of OVA peptide. Data represent 1 of 2 experiments. o indicates EL4 cells defective in GPI anchor biosynthesis; f, EL4 cells with
normal GPI anchor biosynthesis; and *, difference significant (P ⬍ .05) as determined
with the Student t test.
GPIⴚ cells stimulate allogeneic CD4ⴙ T cells less efficiently
than GPIⴙ cells in a proliferation assay
The bm12 mouse has a mutation in the I-Ab␤ gene. CD4⫹ T cells from
bm12 mice are allogeneic to cells bearing wild-type I-Ab molecules.29
We compared the proliferative response of bm12 CD4⫹ T cells to GPI⫺
and GPI⫹ EL4 cells expressing I-Ab (shaded and black bars, respectively, in Figure 4). The response of CD4⫹ T cells was much weaker to
GPI⫺ EL4 than GPI⫹ EL4 cells (2900 versus 8000 cpm in experiment 1
and 3800 versus 17 000 cpm in experiment 2). Therefore, GPI-anchored
proteins on stimulator cells are important for the activation of CD4⫹ T
cells in the allogeneic response.
GPIⴚ multipotential hematopoietic cells could escape CTL
attack in vivo
Based on the result regarding the allogeneic response in vitro, we next
established an in vivo experimental system to compare the susceptibilities of GPI⫹ and GPI⫺ hematopoietic cells to cytotoxic T cells. There is
a report that the transfer of a small number of CD4⫹ T cells from bm12
mice to lightly irradiated B6 mice causes lethal pancytopenia due to a
severe graft-versus-host reaction in bone marrow.27 We transplanted a
mixture of GPI⫹ and GPI⫺ fetal liver cells of B6 background as a source
of hematopoietic stem cells into lethally irradiated B6 mice with or
without CD4⫹ T cells from bm12 mice as a source of CTLs (Figure 5).
To prevent the death of the recipient mice from aplasia, lineage
marker–negative bone marrow cells of bm12 mice were also cotransplanted. The cells derived from fetal liver cells were Ly5.1⫹, whereas
bm12 mice and recipient mice were Ly5.2⫹.
At 5 to 28 weeks after transplantation, we determined percentages of GPI⫺ cells in Ly5.1⫹ cells of various blood cell lineages to
see whether GPI⫺ cells escaped from CTL attack (Figure 6). In
mice without CD4⫹ T cells, Ly5.1⫹ fetal liver–derived cells
dominated in the blood, most likely because syngeneic fetal liver
cells were more efficient than allogeneic bone marrow cells in
engraftment (control mice in Figure 6A). GPI⫺ cells in polymorphonuclear cells (PMNCs) and monocytes numbered about 20%
(control mice in Figure 6B). When CD4⫹ T cells were cotransplanted (PNH model mice in Figure 6A), percentages of fetal
liver–derived cells in PMNCs and monocytes were lower at 5 and 8
weeks (lower than 20% in some mice), indicating that bm12 CD4⫹
T cells killed a large number of B6 fetal liver–derived cells. Under
these conditions, percentages of GPI⫺ cells in PMNCs and
monocytes were markedly increased at 5 and 8 weeks (nearly 100%
RESPONSE OF T CELLS TO GPI ANCHOR–NEGATIVE CELLS
4119
in some mice; Figure 6B-C), indicating that GPI⫹ cells were
selectively killed. In B cells, the contribution of GPI⫺ cells in the
absence of CD4⫹ T cells was lower than in myeloid cells (about
10% versus 20%, control mice in Figure 6B) consistent with our
previous report.11 Nevertheless, in the presence of CD4⫹ T cells,
the percentage of GPI⫺ cells became very high (PNH model mice
in Figure 6B-C) with a decrease of fetal liver–derived B cells
(Figure 6A) at 5 and 8 weeks, again showing selective elimination
of GPI⫹ cells. In CD8⫹ T cells, fetal liver–derived cells appeared
slower than in other cell types (control mice in Figure 6A) but the
percentage of GPI⫺ cells was higher (Figure 6B) consistent with
our report.11 In the presence of allogeneic CD4⫹ T cells, the
percentage of fetal liver–derived CD8⫹ T cells decreased markedly
at 8 weeks (Figure 6A), whereas the percentage of GPI⫺ CD8⫹ T
cells increased (Figure 6B-C). Due to a lack of Ly5 expression,
erythrocytes derived from B6 fetal liver cannot be distinguished
from those derived from bm12 bone marrow. The percentage of
GPI⫺ erythrocytes in the absence of CD4⫹ T cells was 10% to 40%
at 5 to 14 weeks (control mice in Figure 6B). It increased in some
mice in the presence of CD4⫹ T cells, however, not significantly
(Figure 6B-C). These results indicate that GPI⫺ multipotential
hematopoietic cells are more resistant to CTL attack.
In mice that received cotransplantations with CD4⫹ T cells,
percentages of fetal liver–derived cells increased with time (Figure
6A) in association with a decrease in the percentage of GPI⫺ cells
(Figure 6B). Both percentages were similar to those in the control
mice without bm12 CD4⫹ T cells at 21 weeks after transplantation.
Therefore, GPI⫹ multipotential hematopoietic stem cells of fetal
livers were not eliminated by bm12 CD4⫹ T cells and on decline of
the allogeneic CD4⫹ T cells provided GPI⫹ progeny in blood. This
indicates that multipotential hematopoietic progenitors but not
stem cells express MHC class II I-A molecules and are sensitive to
allogeneic CD4⫹ T cells. This again indicates that expansion of the
GPI⫺ cell population occurred only under selection by allogenic T
cells. The results in this mouse model support the idea that if
autoimmune cytotoxic CD4⫹ T cells recognize multipotential
hematopoietic cells, GPI⫺ cells would survive and expand.
Discussion
This study presents the first experimental evidence that supports
the immunologic selection hypothesis for the clonal expansion of
Figure 4. GPIⴚ EL4 cells were less efficient in proliferative activation of
allogeneic CD4ⴙ T cells than GPIⴙ EL4 cells. Purified CD4⫹ T cells from a bm12
mouse were incubated with GPI⫺ (shaded) and GPI⫹ (black), I-Ab–expressing EL4
cells. Proliferation was assessed on day 4. Two similar experiments are shown;
o indicates GPI⫺; f, GPI⫹; and * indicates difference significant (P ⬍ .05).
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Figure 5. In vivo experimental system to test resistance of
GPI anchor–defective hematopoietic cells to cytotoxic cells.
Pigaflox males were crossed with hCMV-Cre transgenic females.
Female embryos that had the Cre transgene became heterozygous for the Piga disruption and mosaic for Piga expression due
to random X inactivation. Fetal liver cells of the mosaic embryos
on 14 dac were transferred to lethally irradiated B6 recipients
together with lineage marker–negative bone marrow cells of
bm12 mice, with or without purified CD4⫹ T cells from bm12 mice.
To distinguish fetal liver–derived cells from recipient-derived and
bm12 bone marrow–derived cells, mice with the Ly5.1 allele were
used as a source of fetal livers and mice with the Ly5.2 allele were
used for the recipients and as a source of bone marrow.
PNH cells. Especially, it was demonstrated that GPI⫺ hematopoietic cells become dominant in model mice on selection by
allogeneic CD4⫹ T cells (Figure 6). Until now there has been only
clinical evidence for the immunologic selection hypothesis. The
frequent association of PNH with AA,30,31 taken together with the
presence of PIG-A mutant blood cells in most healthy individuals,13 has
Figure 6. GPIⴚ multipotential hematopoietic cells can
escape CTL attack. (A) Percentages of Ly5.1⫹ fetal
liver–derived cells in various blood cells of individual mice
were shown as a function of weeks after transplantation.
Upper panels, control mice (n ⫽ 7) that received transplants of GPI⫹ and GPI⫺ fetal liver cells, and lineage
marker–negative bm12 bone marrow cells; lower panels,
experimental mice (n ⫽ 9) that received transplants of
fetal liver cells and lineage marker–negative bm12 bone
marrow cells together with CD4⫹ T cells from bm12 mice.
(B) Percentages of GPI⫺ cells in fetal liver–derived cells
of various blood cell types plotted as a function of weeks
after transplantation. Control and experimental mice
were the same as those in panel A. (C) Percentages of
GPI⫺ cells in various blood cell lineages determined 5
and 8 weeks after transplantation are shown (data for
PMNCs and monocytes are at 5 weeks and for B and T
cells and erythrocytes are at 8 weeks). E indicates
control mice (n ⫽ 7); F, experimental mice (n ⫽ 9). Bars
represent mean percentages.
been the basis of the suggestion that an autoimmune process promotes
clonal expansion. We assumed CD4⫹ T cells to be cytotoxic effectors
and established 3 experimental systems to test 2 hypothetical mechanisms of the immunologic selection (Figure 1). First, we tested a
specialized situation in which the antigen recognized by CD4⫹ T cells is
derived from GPI-anchored protein (Figure 1A) and showed that GPI⫹
BLOOD, 1 DECEMBER 2002 䡠 VOLUME 100, NUMBER 12
APCs present the antigen derived from GPI-anchored proteins on MHC
class II molecules, whereas GPI⫺ APCs do not (Figure 2). GPI-anchored
proteins like other cell surface transmembrane proteins would reach, via
the endocytic pathway, a compartment where proteins are processed for
presentation on MHC class II molecules. Proteins normally GPI
anchored are not expressed on the surface of GPI⫺ APCs; hence, there is
no presentation on the MHC class II molecules. Our data suggest that if
the relevant autoantigen is derived from GPI-anchored proteins, PNH
cells may be resistant to CD4⫹ CTL (Figure 1A).
Second, we demonstrated using in vitro systems that GPI⫺
APCs do not stimulate antigen-specific and allogeneic CD4⫹ T
cells efficiently (Figures 3 and 4). We think that some GPIanchored protein on APCs acts as a ligand for costimulatory
molecules on T cells (Figure 1B). A candidate molecule is CD48
that is expressed on the surface of APCs and interacts with CD2 on
T cells. An experiment using CD48 knockout mice revealed that the
alloresponse of CD4⫹ T cells was significantly lower without
CD48 on the surface of APCs.32 Indeed, GPI⫹ EL4 cells used as
model APCs in our experiments expressed CD48, whereas GPI⫺ EL4
cells did not (data not shown). Because CD2 is widely expressed on T
cells, costimulation mediated by interaction of CD48 and CD2 would be
involved in our in vitro experimental systems.
Third, using allogeneic CD4⫹ T cells in a transplantation system, we
demonstrated the expansion of the GPI⫺ hematopoietic cell population
in vivo on selection by T cells (Figure 6). In this system, the expansion
was seen only transiently. Both GPI⫹ and GPI⫺ multipotential hematopoietic stem cells were spared and when allogeneic T cells declined, the
dominance of GPI⫺ cells disappeared. It is most likely that the mouse
stem cells derived from fetal liver do not express MHC class II I-A
molecules and the selection occurred in the multipotential hematopoietic
progenitors that presumably express I-A⫹.27 In this regard, this mouse
model is different from AA where multipotential hematopoietic stem
cells are a target of autoimmunity. However, it is likely that MHC class
II molecules are expressed on the multipotential hematopoietic stem
cells in patients with AA because interferon ␥, which induces MHC
class II expression in various cells,33 is expressed in cells from patients
with AA.28 Also, there is a report that human multipotential hematopoietic stem cells express HLA-DR.34 Therefore, the multipotential
hematopoietic stem cells could be recognized by CD4⫹ T cells in a
human system.
As shown in our mouse system, expansion of the GPI⫺
population due to selection at the progenitor level is reversible if
the stem cells are spared; when the CTLs are present, GPI⫺ cells
would continuously proliferate and when CTLs disappear, the
expansion would also stop. It is possible that some of the reported
patients with PNH with spontaneous remission were mediated by a
similar mechanism.35 The candidate GPI-anchored protein responsible for the selection in our mouse model would be CD48 because
Blazar and colleagues reported that when bm12 CD4⫹ T cells were
RESPONSE OF T CELLS TO GPI ANCHOR–NEGATIVE CELLS
4121
transplanted into lightly irradiated B6 mice, the death of recipient mice
due to aplasia was nearly completely prevented by infusion of antiCD48 mAbs.36 Human CD48 is also GPI anchored. Although human
CD48 does not bind CD2 with high affinity, it binds to CD244 2B4, a
recently identified costimulatory molecule on T cells.37,38
The PNH cells are present in a large proportion of AA patients.39,40
Although the percentage of GPI⫺ cells is higher in AA patients than
healthy individuals, the cells are frequently stable and do not reach a size
big enough to cause the clinical manifestation of PNH.39 GPI⫺ cells
stimulate T cells less efficiently, but the stimulation is not completely
abolished. It is possible that under strong marrow-damaging conditions,
expansion may be limited. When the patients are treated with immunosuppressive therapies, such as antithymocyte globulin or cyclosporin A,
and if the therapeutic effect is partial, GPI⫺ cells may proliferate
selectively, resulting in the development of PNH. If the therapy is very
effective, selection may disappear, leading to the growth of GPI⫹ cells
and remission of AA.40
We are not proposing that selection is the only mechanism of
clonal expansion in PNH. The 2 hypotheses (see “Introduction”)
are not mutually exclusive and both may be operating. We think
that selection may not be sufficient for high-level expansion of the
PIG-A mutant clone seen in so-called florid PNH. Such a clone may
have a growth phenotype, like benign tumors, owing to additional
cytogenetic abnormalities. Consistent with this idea, there is a
recent report that the early growth response gene (EGR1), a zinc
finger transcription factor, is up-regulated in all cases of PNH
studied.41 When immunologic damage occurs in a pool of hematopoietic stem cells, the surviving GPI⫺ stem cells would be forced to
proliferate. Then the possibility of a second genetic abnormality
increases, resulting in the generation of a subclone having a growth
phenotype. Up-regulation of EGR1 may be due to a genetic
abnormality other than the PIGA somatic mutation. It is well known
that some patients with PNH eventually develop acute myelogenous
leukemia from within the PNH clone.6 It is conceivable that the further
accumulation of genetic abnormalities in a benign tumorlike subclone
leads to the generation of a leukemic clone.
The identification of GPI-anchored proteins responsible for the
immunologic selection, the demonstration of the target protein that is
recognized by autoreactive CTLs in PNH, and the search for additional
cytogenetic abnormalities responsible for the growth phenotype are
critical to further understand clonal expansion in PNH.
Acknowledgments
We thank Drs F. R. Carbone, H. Nakauchi, T. Sato, L. Karlsson, T.
Seya and T. Kina for their gifts, Dr W. Hazenbos for discussion, Dr
Shuichi Yamada for generating transgenic mice, and Keiko Kinoshita and Fumiko Ishii for technical assistance.
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