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RED CELLS
From the Second Department of Internal Medicine,
Kumamoto University School of Medicine, Japan; Hematology Branch,
National Heart, Lung, and Blood Institute, National Institutes of
Health, Bethesda, MD; Department of Immunoregulation, Research
Institute for Microbial Diseases, Osaka University, Japan; Department
of Biochemistry and Applied Biosciences, Miyazaki University, Japan.
The cloning of the PIG-A gene has facilitated the
unraveling of the complex pathophysiology of paroxysmal nocturnal
hemoglobinuria (PNH). Of current major concern is the mechanism by
which a PNH clone expands. Many reports have suggested that an immune
mechanism operates to cause bone marrow failure in some patients with
PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow
diseases, one hypothesis is that the PNH clone escapes immune attack,
producing a survival advantage by immunoselection. To test this
hypothesis, we examined the sensitivity of blood cells, with or without
PIG-A mutations, to killing by natural killer (NK) cells,
using 51Cr-release assay in vitro. To both peripheral blood
and cultured NK cells, PIG-A mutant cells prepared from
myeloid and lymphoid leukemic cell lines were less susceptible than
their control counterparts (reverted from the mutant cells by
transfection with a PIG-A cDNA). NK activity was completely
abolished with concanamycin A and by calcium chelation, indicating that
killing was perforin-dependent. There were no differences in major
histocompatibility (MHC) class I expression or sensitivity to either
purified perforin or to interleukin-2-activated NK cells between
PIG-A mutant and control cells. From these results, we
infer that PIG-A mutant cells lack molecules needed for NK
activation or to trigger perforin-mediated killing. Our experiments
suggest that PIG-A mutations confer a relative survival
advantage to a PNH clone, contributing to selective expansion of these
cells in the setting of marrow injury by cytotoxic lymphocytes.
(Blood. 2002;100:1031-1037) Paroxysmal nocturnal hemoglobinuria (PNH) is an
acquired, clonal stem cell disorder that manifests clinically as
intravascular hemolysis, venous thrombosis, frequent episodes of
infection, and rare leukemic conversion.1,2 In addition,
PNH is often a bone marrow (BM) failure syndrome, strongly related to
aplastic anemia and the myelodysplastic syndromes
(MDSs).3,4 All 3 processes may occur in a single
patient.5,6 Among the clinical manifestations, the
molecular events leading to hemolysis have been
elucidated7-9 and the responsible gene PIG-A
has been cloned.10 PNH cells with PIG-A
mutation do not produce glycosylphosphatidylinositol (GPI) and lack
cell surface expression of a large family of proteins that utilize GPI
to localize in the plasma membrane.10 Therefore, PNH
cells, deficient in complement regulatory GPI-linked proteins such as
decay-accelerating factor (DAF) and CD59, undergo complement-mediated hemolysis characteristic of PNH.11 A mechanism of
infection-associated precipitation of hemolysis also has been
shown.12 Proclivity to thrombosis is also attributable to
PIG-A mutations, but a molecular mechanism has not been
found.13,14
Despite rapid progress in understanding PNH pathophysiology, the
etiology and the mechanism by which a PNH clone expands are still
unknown.15 Murine pig-a knockout models have
shown that clonal expansion of PNH cells does not follow on the
presence of the gene mutation alone.16-18 Furthermore,
small numbers of PIG-A mutant cells are present in healthy
individuals.19,20 Alternative theories have been
proposed.15,21,22 An intrinsic alteration might confer a
proliferative advantage to PNH cells over normal cells, according to
one growth advantage hypothesis. Leukemic conversion can occur in PNH
patients, albeit infrequently.6,23 A predominant
proliferating clone is often detected among multiple PNH clones within
the same patient.24-26 Transplantation experiments in mice
have suggested an intrinsic growth alteration in PNH stem cells.22 However, the gene responsible for such behavior
is not known.
Alternatively, the lack of GPI-linked membrane proteins might confer a
relative survival advantage to PNH clones.15,21 There is
evidence for15,27 and against such a survival
advantage.28 For example, it is widely accepted that an
immune-mediated BM injury occurs in patients with
PNH,29-32 aplastic anemia,33,34 and
MDS,35 and blood cells with PIG-A mutation (or
PNH phenotype) are detected more abundantly in patients with BM failure
syndromes than in healthy individuals.4,36-38 A PNH clone
could escape immune-mediated BM attack and survive preferentially in
such a pathologic environment. Lymphocytes of PNH phenotype appeared in
patients with non-Hodgkin lymphoma after therapy with antibodies to the
GPI-linked lymphocyte antigen CD52, an example of iatrogenic immunoselection of PIG-A mutant cells.39,40 To
date, the incitement and molecular events in the immune attack to BM
cells by cytotoxic lymphocytes are unclear. As candidates for the
pathogenic effector cells, T cells34 and natural killer
(NK) cells41,42 have been most studied. Involvement of NK
cells in BM impairment is suggested by clinical
observation43 and experimental data.44 Recent
evidence has indicated a close relationship between NK cells (serving
innate immunity) and cytotoxic T lymphocytes (acquired immunity).45-47 In this context, and in order to develop
evidence relating to the survival advantage theory, we prepared
leukemic cells with PIG-A mutations and their control
counterparts by transfection with a PIG-A cDNA, and then
compared their sensitivity to killing by NK cells in vitro.
Preparation of target cells with and without PIG-A
mutations
Preparation of NK cells as cytotoxic effector lymphocytes
Flow cytometry
Cytotoxicity assay Natural killer activity was determined by 51Cr-release assay as described elsewhere.28,52 In brief, the number of target cells with and without PIG-A mutation was accurately adjusted to 5 × 105/mL with polystyrene fluorospheres (Flow-Count; Beckman Coulter) and a flow cytometer (Epics).53 Target cells (1 mL) were incubated with 3.7 MBq of Na2 51CrO4 (New England Nuclear, Boston, MA) for 3 hours at 37°C, 5% CO2, and washed twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) (Sigma, St Louis, MO). After 10-fold dilution, target cells (5 × 103/100 µL per well) were mixed with NK cells at various ratios of effector and target cells (E/T ratio) in U-bottomed 96-well microtiter plates (Corning, New York, NY) in triplicate. Cells were then incubated for 3 hours at 37°C, 5% CO2 in RPMI 1640 supplemented with 10% FCS and 10mM HEPES (Sigma) at pH 7.2. Medium containing 51Cr liberated from target cells was collected using cellulose acetate absorption cartridges (Skatron, Lier, Norway). Radioactivity was determined with automated -counter
(Minaxi 5530; Packard Instrument Company, Downers Grove, IL). There was
no difference in the uptake and spontaneous release of 51Cr
between target cells with and without PIG-A mutation (data not shown). Spontaneous 51Cr release was determined by
adding culture medium instead of effector cells to target cells.
Maximum release was determined by adding Triton X-100 (0.25% final
concentration; Nacalai Tesque, Kyoto, Japan) to the target cells.
Spontaneous release was less than 10% of maximum release. Percent
cytotoxicity was calculated using the following formula: % specific
cytotoxicity = (experimental release  spontaneous
release) / (maximum release spontaneous release) × 100.
Next, we performed the cytotoxicity assay under the coexistence of PIG-A mutant and control cells to mimic PNH patients' BM where blood cells with and without PIG-A mutation coexist. In brief, we prepared target cells by mixing the same number of PIG-A mutant and control K562 cells in the same well. Either the mutant or control cells in the mixture were labeled with 51Cr to assess the selection of target cells in the killing. Cells were then incubated with NK cells (2E3) at an E/T ratio (2:1) and cytotoxicity was assessed as described above. We determined the sensitivity of target cells with and without PIG-A mutation to perforin. Perforin was purified from a mouse cytotoxic lymphocyte cell line (CTLL-R8.6) as reported previously.54,55 In brief, cells were disrupted using a nitrogen cavitation bomb in the presence of protease inhibitors. From the cell lysate, perforin-containing granules were collected by Percoll (Sigma) gradient centrifugation. Perforin was extracted from the granules by homogenization and purified by immobilized metal affinity column chromatography and hydrophobic interaction chromatography. Perforin activity was determined by cytolysis of sheep erythrocytes (1 unit corresponds to 50% hemolysis of 2 × 107 cells/200 µL). Cytotoxicity was determined by incubation of 6 U purified perforin with target cells for 1 hour in the absence of FCS. Other conditions were the same as those used for cytotoxic lymphocytes. (Mouse perforin exerts cytotoxicity to human cells as well as to mouse cells.56,57) Inhibition of cytotoxicity To exert cytotoxicity, lymphocytes usually use perforin/granzyme, Fas/Fas ligand, and tumor necrosis factor (TNF).58-60 Perforin-mediated cytotoxicity is inhibited with concanamycin A (CMA; Wako, Osaka, Japan) and a calcium chelate, ethylene glycol bis ( -aminoethylether)-N, N, N', N'-tetraacetic acid
(EGTA; Nacalai Tesque). CMA, a specific inhibitor of vacuolar type
H+-adenosine triphosphatase (ATPase), accelerates
degradation of perforin by an increase in the pH of lytic
granules.61 To inhibit perforin-mediated cytotoxicity,
killing was performed in the presence of EGTA or CMA at various
concentrations. CMA required pretreatment of effector cells for 2 hours.
To determine the role of GPI-anchored proteins such as DAF and CD59 that are expressed only on the control cells, we tested cytotoxicity in the presence of 10 µg/mL MoAb to DAF or CD59. MoAbs to DAF were 1A10, BRIC 216 (mouse IgG1; Ylem, Roma, Italy), 1C6 (mouse IgG1; Wako), and MEM-118 (mouse IgM; Monosan, Uden, The Netherlands). MoAbs to CD59 were MEM-43 (mouse IgG2a; Monosan), MEM-43/5 (mouse IgG2b; Monosan), MEM-125 (mouse IgM; Monosan), 5H8, 2/24 (mouse IgG1; Chemicon, Temecula, CA), p282 (mouse IgG2a; Pharmingen), YTH53.1 (Rat IgG2b; Serotec, Oxford, United Kingdom), and BRA10G (mouse IgG1; Ancell, Bayport, MN). Isotype-matched control Ig was obtained from Beckman Coulter. Determination of cytokines by enzyme-linked immunosorbent assay To characterize the activation of NK cells, we measured the concentrations of interferon (IFN-) and TNF- released from NK
cells in the coculture with target cells. We used peripheral blood NK
cells isolated from 5 healthy volunteers. Targets were PIG-A
mutant and control K562 cells. NK and target cells were incubated at an
E/T ratio (32:1) for up to 12 hours. Cell culture supernatant was
tested for cytokines using sandwich enzyme-linked immunoabsorbent assay
(ELISA) kits (R&D Systems, Minneapolis, MN) according to the
manufacturer's instructions.62 Briefly, wells in a
microtiter plate were coated with antibody specific for IFN- or
TNF-, incubated with standards or samples, labeled with an
enzyme-linked antibody specific for each cytokine, and incubated with a
substrate solution. The color developed and the intensity of the color
was measured at 450 nm. Each assay was performed in duplicate. The
minimum detectable doses of IFN- and TNF- were less than 8 pg/mL
and 4.4 pg/mL, respectively.
Analysis of perforin mRNA expression by reverse transcription-polymerase chain reaction We performed reverse transcription-polymerase chain reaction (RT-PCR) to analyze the expression of perforin mRNA in peripheral blood NK cells obtained from 2 healthy donors, as described.63,64 After a 3-hour coculture with target K562 cells (E/T ratio, 32:1), NK cells were collected with magnetic beads coated with MoAb to CD2. From the NK cells, total RNA was isolated using a kit with a silica-gel membrane (RNeasy; Qiagen, Hilden, Germany) and converted to cDNA with a kit (ReverTra; Toyobo, Osaka, Japan) according to the manufacturer's instructions.64 The coding region of the cDNA was then amplified by PCR with 0.5 U recombinant Taq DNA polymerase (ExTaq; Takara, Shiga, Japan), 200 µM dNTPs in a buffer (25 µL; 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin), and 10 pmol of a sense primer (5'-GAGGCCCAGGTCAACATAGGCA-3') and an antisense primer (5'-TCACCACACGGCCCCACTCCG-3').63 After initial denaturation at 94°C for 5 minutes, DNA amplification was performed with a PCR thermocycler (Astec, Fukuoka, Japan) for 30 cycles; each cycle consisted of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes with a final extention at 72°C for 10 minutes. PCR products were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining.Statistical analysis Student paired t test was used to determine significance levels. P values less than .05 were considered significant.
Decreased susceptibility of PIG-A mutant to NK cells K562 cells with PIG-A mutation were less susceptible to NK cell killing than were control counterparts with a functional PIG-A gene (Figure 2A). To confirm decreased sensitivity of the PIG-A mutant, we performed additional experiments using various effector (NK) and target cells. First we repeated the cytotoxicity assay using peripheral NK cells isolated from different individual healthy donors (Figure 2B). Each experiment showed decreased sensitivity of PIG-A mutant K562 cells. When cytolysis of control cells was normalized to 100%, cytolysis of PIG-A mutant cells ranged from 36% to 90% (mean 70%). The difference in lysis of K562 cells with and without a PIG-A mutation was significant (Student paired t test, P < .000 01). Killing of our K562 cells was less than that of wild-type K562 cells (data not shown), probably due to the weak expression of MHC class I in our line inhibiting NK activity (Figure 1B).65 MHC expression on the target cells may also explain the variation in their killing by peripheral NK cells from the different healthy donors (Figure 2B).66 Further experiments, using a cultured NK cell line (2E3) (Figure 3A,C) instead of peripheral NK cells or using lymphoid leukemic cell lines (B, JY-5; and T, MOLT-4) instead of K562 (Figure 3B-C), also showed decreased sensitivity of PIG-A mutant to NK cells. Moreover, the decreased susceptibility of mutant cells to NK cells could also be observed in cytotoxicity assays combining mutant and control cells (Figure 4).
Perforin-mediated cytotoxicity of NK cells Because NK cells (2E3) exert cytotoxicity despite their lack of Fas ligand (Figure 3A,C), we first examined the effects of CMA and EGTA, which inhibit the perforin/granzyme pathway. Each reagent inhibited dose-dependently the killing of K562 cells with and without PIG-A mutation by either peripheral (Figure 5) or cultured NK cells (data not shown). Killing was virtually abolished with either 5 nM of CMA or 1 mM of EGTA, and at these concentrations NK cells remained viable. These findings suggested that NK cells used only the perforin/granzyme pathway. Next, we replaced NK cells with purified perforin in the cytotoxicity assay (Figure 6). In the presence of soluble perforin, both mutant K562 and JY-5 cells were lysed similar to controls, indicating that the PIG-A mutation did not decrease sensitivity to perforin. There was a difference in susceptibility to perforin between K562 and JY-5.
Defective activation of NK cells by PIG-A mutant cells Figure 7 shows the 3-hour cytotoxicity assay in the presence of 10 ng/mL IL-2, a potent activator of NK cells. IL-2 enhanced the target cell (K562) killing by NK cells isolated from 3 healthy volunteers but diminished or abolished the difference in the sensitivity to killing between PIG-A mutant and control cells (Figure 7A-C). The mutant and control cells thus showed similar sensitivity to activated NK cells (Figure 7) as well as to purified soluble perforin (Figure 6). Additionally, killing was completely suppressed with perforin inhibitors (data not shown). We inferred that NK cells, once sufficiently activated with IL-2 to release perforin/granzyme, were equally effective against PIG-A mutant and control cells.
We also measured IFN-
To further explore the possibility of impaired triggering of the
perforin/granzyme pathway by PIG-A mutant cells, we screened GPI-linked membrane molecules missing from the mutant cells by flow
cytometry. However, no MoAb clones to DAF (data not shown) and CD59
(Figure 9) exerted any effects in
cytotoxicity assays with cultured NK cells. In these experiments,
antibody-dependent cellular cytotoxicity was negligible because
cultured NK cells lacked CD16 (Fc
Little is known about the mechanism responsible for expansion of
PNH clones.15,21,22 The present work supports a selective survival advantage theory. In our experiments, leukemic cells with
PIG-A mutations (GPI We investigated the mechanism for PIG-A mutant insensitivity
to perforin-mediated killing by NK cells in their coculture. In
general, perforin-mediated killing by NK cells consists of at least 3 discrete steps: first, stimulation of NK cells by direct contact
between NK and target cells; second, signal transduction in NK cells to
release perforin/granzyme; third, killing targets cells with the
liberated cytotoxic molecules.69 In this regard, the
PIG-A mutant and control cells showed similar sensitivity to
soluble perforin used instead of NK cells (consistent with previous
report67) or to perforin-mediated killing by NK cells driven by IL-2. Thus, our findings indicate that these later steps are
not responsible for the difference in NK sensitivity between target
cells. Rather, by exclusion, the first event appears important: resistance is manifest when NK cells are activated by contact with
PIG-A mutant cells. It is thus conceivable that
PIG-A mutant cells are partly defective in their ability to
signal the release of perforin (although we did not determine the
relative concentrations of perforin liberated in these experiments). In
further characterization of NK activation, coculture of NK cells with
target cells induced marked release of IFN- NK cell activation by contact with target cells is mediated or inhibited by cell surface molecules. PIG-A mutant cells distinctly lack GPI-linked membrane proteins. Although inhibition assays with MoAbs to DAF and CD59 gave negative results, unidentified GPI-linked proteins may explain the decreased sensitivity of PIG-A mutant to NK cells. To date, 2 GPI-linked glycoproteins (mouse RAE-1 and human ULBP) have been shown to activate NK cells and to be inducible by transformation and viral infection.70-72 Of interest, such activation signals are dominant over inhibitory signals from MHC class I.72,73 NK cells killed leukemic cells despite their MHC class I expression in our assay. The identification of such putative membrane molecules suggested in the present study could provide a new strategy to control NK activity and also facilitate the unraveling of NK cell-associated pathophysiology of infection, cancer, autoimmune disease, and transplantation.60,74-76
We thank Masato Yagita of Osaka Kitano Hospital, Jaroslaw Maciejewski of National Institutes of Health, and Yasuharu Nishimura and Masafumi Takiguchi of Kumamoto University for their advice and help.
Submitted November 7, 2001; accepted March 4, 2002.
Supported by a grant from the Sagawa Foundation for Promotion of Cancer Research, and grants for scientific research and for cancer research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
S.N. and S. I. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hideki Nakakuma, Second Department of Internal Medicine, Kumamoto University, School of Medicine, Honjo 1-1-1, Kumamoto 860-8556, Japan; e-mail: nakakuma{at}kaiju.medic.kumamoto-u.ac.jp.
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  C. Sugimori, T. Chuhjo, X. Feng, H. Yamazaki, A. Takami, M. Teramura, H. Mizoguchi, M. Omine, and S. Nakao Minor population of CD55-CD59- blood cells predicts response to immunosuppressive therapy and prognosis in patients with aplastic anemia Blood, February 15, 2006; 107(4): 1308 - 1314. [Abstract] [Full Text] [PDF]
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N. Hanaoka, T. Kawaguchi, K. Horikawa, S. Nagakura, H. Mitsuya, and H. Nakakuma Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP Blood, February 1, 2006; 107(3): 1184 - 1191. [Abstract] [Full Text] [PDF]
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Y. Takahashi, J. P. McCoy Jr, C. Carvallo, C. Rivera, T. Igarashi, R. Srinivasan, N. S. Young, and R. W. Childs In vitro and in vivo evidence of PNH cell sensitivity to immune attack after nonmyeloablative allogeneic hematopoietic cell transplantation Blood, February 15, 2004; 103(4): 1383 - 1390. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
cells but appeared on the control counterparts
(GPI+ cells) (A). The pair of the mutant
(GPI
), control cells
(revertant) expressing GPI; and GPI
),
PIG-A mutant cells (mock transfectant) deficient in GPI. (A)
Cytolysis by NK cells obtained from a representative healthy donor at
various E/T ratios. (B) Relative decrease in the cytolysis of
GPI
) and without
(
) PIG-A mutation in the presence of a calcium chelate
EGTA (A) and concanamycin A (B). E/T ratio was 64:1. Each value
represents the mean (± SD) of triplicate assays.
