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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1799-1806
Lymphocyte Subset Analysis and Glycosylphosphatidylinositol
Phenotype in Patients With Paroxysmal Nocturnal Hemoglobinuria
By
Stephen. J. Richards,
Derek. R. Norfolk,
David M. Swirsky, and
Peter Hillmen
From the Haematological Malignancy Diagnostic Service, Leeds General
Infirmary, Leeds; and the Department of Haematology, Imperial College
School of Medicine, The Hammersmith Hospital, London, UK.
 |
ABSTRACT |
Using multicolor flow-cytometry we have examined 19 patients with
paroxysmal nocturnal hemoglobinuria (PNH) (18 with active disease and 1 spontaneous remitter) to determine absolute numbers of lymphocyte
subsets and the proportion of glycosylphosphatidylinositol (GPI)-deficient clones amongst these subpopulations. Lymphocyte subsets
were abnormal in all patients; the most frequent findings were low
absolute numbers of natural killer (NK) cells (median, 0.08 × 109/L; normal range, 0.2 to 0.4 × 109/L) and
low absolute numbers of B cells (median, 0.05 × 109/L;
normal range, 0.06 to 0.65 × 109/L). GPI-deficient B, T,
and NK cells were identified in 88%, 84%, and 89% of patients,
respectively. The proportion of GPI-deficient cells within individual
lymphoid lineages was highly variable, though in most patients the
percentage of GPI-deficient NK cells was considerably higher than B or
T cells. These observations can be explained when mechanisms of normal
lymphopoiesis are considered. Despite these quantitative and
qualitative abnormalities, no patients suffered an excessive number or
severity of infections. The detection of PNH clones amongst all
lymphocyte lineages may provide important information regarding the
natural history of the disease and additional insights into kinetics of
adult lymphopoiesis.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
PAROXYSMAL NOCTURNAL hemoglobinuria (PNH)
is an acquired hematopoietic stem cell disorder in which a somatic
mutation of the X-linked PIG-A gene results in a partial or
absolute deficiency of all proteins linked to the cell membrane by the
glycosylphosphatidylinositol (GPI) anchor.1-6 Most, if not
all, patients with PNH have evidence of underlying bone marrow failure.
In addition, up to 50% of patients with aplastic anemia go on to
develop a PNH clone. Thus, there is a very close association between
aplastic anemia and PNH. The successful use of immunosuppressive
therapy such as antithymocyte globulin (ATG) for the treatment and
control of aplastic anemia provides strong evidence that there may be
an immunological component to the disease, and it appears that this
promotes the development of PNH clones.7,8 The most
profound clinical feature of PNH is a hemolytic anemia due to a
deficiency from the red blood cell (RBC) membrane of the complement
regulatory molecules CD55 (decay accelerating factor [DAF]) and CD59
(membrane inhibitor of reactive lysis [MIRL]). This pathobiological
property renders erythrocytes abnormally sensitive to the lytic action
of complement and was exploited as the basis for the acidified serum
lysis (Ham) test for the diagnosis of PNH.9-12 This test
has now largely been superseded by flow cytometry as the standard
diagnostic test for PNH.13,14 The use of flow cytometry and
specific combinations of monoclonal antibodies has enhanced the ability
to detect small PNH clones within multiple hematopoietic cell lineages.
Consequently, this has led to reclassifying PNH into two types: (1)
hemolytic PNH, characterized by overt episodes of hemolytic anemia, and
(2) hypoplastic PNH in which clones of GPI-deficient hematopoietic
cells are detectable with no overt hemolysis but may have other
symptoms such as aplastic anemia, leukopenia, or thrombocytopenia.
In PNH the presence of GPI-deficient clones within the platelet,
monocyte, and granulocyte components of the myeloid lineage is well
documented, and a small number of investigators have also unequivocally
described PNH clones within peripheral blood lymphocyte populations.13,15-20 Indeed, GPI-deficient T-lymphocyte
populations have been described in two patients in long-term clinical
remission for PNH with no detectable granulocyte or red cell
clones.21,22 Evidence for the presence of cells derived
from the PNH clone within the natural killer (NK) cell lineage is less
clear, mainly due to the fact that there is no single pan-NK-cell
antigen analogous to CD3 and CD19 that defines T- and B-lymphocyte
populations, respectively. The majority of NK cells can be identified
phenotypically on a multiparameter basis by concomitant expression of
CD16 and CD56 membrane determinants with a lack of CD3 or by a
CD3 CD7+ composite
phenotype.23,24 To identify precisely PNH clones within the
NK-cell population, three-color flow cytometry is a minimum
requirement.
Lymphopenia is a well-recognized feature of PNH, although few authors
have examined lymphocyte subset distributions and absolute numbers in
any detail. In this present study, multicolor flow cytometry is used to
show that lymphocyte subset abnormalities are a common feature of PNH
and to present unequivocal evidence that all lymphoid lineages may
contain distinct PNH clones.
| |
MATERIALS AND METHODS |
Patients studied.
After obtaining informed consent, peripheral blood samples were
collected from 19 adult patients with PNH (including 1 patient who
showed spontaneous remission 15 years ago) and 5 healthy adult normal
controls. Patients who showed clearly demonstrable evidence of
macroscopic hemoglobinuria were classified as hemolytic, and those
without were classified as hypoplastic. The patient group comprized 8 males and 11 females, with an age range of 22 to 66 years. Full blood
counts were performed on a Sysmex K1000 cell counter (Sysmex,
Milton Keynes, UK). The initial diagnosis of PNH was
made by flow cytometry, showing the absence of GPI-linked proteins from
red cell and/or granulocyte surface membranes as described
elsewhere or by a positive Ham test.20 Lymphocyte subpopulation numbers were determined on all patient samples by flow
cytometry. GPI-linked antigen expression by lymphocytes was studied on
all patient samples by a whole blood lysis technique and also by
lymphocytes isolated by density sedimentation technique.
Lymphocyte subsets.
An initial whole blood lysis screen was performed to assess the
proportions and absolute numbers of major lymphocyte populations (B, T,
and NK cells), T-cell subsets, and activated T cells. Briefly, 30-µL
volumes of the following combinations of monoclonal antibodies (fluorescein isothiocyanate [FITC]/phycoerythrin
[PE] or FITC/PE/Cy5) were pipetted into five 12 × 75-mm
polystyrene tubes (Becton Dickinson, San Jose, CA):
CD45/CD14; control; CD4/CD8/CD3; CD3/HLA-DR/CD19; and CD16/CD56/CD3.
One hundred microliter volumes of EDTA anticoagulated whole blood were
then added, mixed, and incubated in the dark at ambient temperature for
20 minutes and punctuated by gentle mixing every 5 minutes. A 2-mL
volume of a 1/10 dilution of fluorescence-activated cell sorter (FACS)
lysing solution (Becton Dickinson) was added to each tube, gently
mixed, and incubated for a further 10 minutes in the dark. The tubes
were then centrifuged at 300g for 90 seconds, the supernatant
discarded, and the white cell pellet resuspended in a 2-mL volume of
FACSFlow (Becton Dickinson) supplemented with 0.5% bovine serum
albumin (BSA; Sigma Chemicals, St Louis, MO). The cells
were again pelleted and washed with a further 2-mL volume of
FACSFlow/BSA. Following this final wash stage, the cells were resuspended in a 300-µL volume of a 1/10 dilution of CellFIX (Becton Dickinson) and incubated for a minimum of 5 minutes before flow cytometry analysis. The cells were then analyzed using a FACScan flow
cytometer (Becton Dickinson) and Lysis II software
(Becton Dickinson) as previously described collecting a minimum of
10,000 gated lymphocyte events.24 Control studies comprised
a CD45/CD14 Leucogate (Becton Dickinson)-stained sample, and cells
stained with irrelevant isotype matched negative control antibodies.
The sources and specificities of monoclonal antibodies used for
immunophenotyping are summarized in Table
1. Absolute numbers of lymphocyte subsets were calculated using the
Sysmex lymphocyte count multiplied by the percentage of lymphocytes
expressing individual CD determinants divided by 100.
Separation of mononuclear cells.
Mononuclear cells were fractionated from 10-mL volumes of EDTA
anticoagulated blood by conventional density sedimentation with
Lymphoprep (Nycomed, Oslo, Norway). Cells collected
from the plasma/lymphoprep interface were washed twice with 10-mL
volumes of FACSFlow/BSA, and the white blood cell count was adjusted to 50 × 109/L for subsequent immunophenotyping studies.
Immunophenotyping and flow cytometry of mononuclear cells.
Ten-microliter volumes of cells were stained in U-shaped 96-well
microtiter plates with the following two- and three-color combinations
of monoclonal antibodies (FITC/Cy5 and FITC/PE/Cy5): CD59/CD3,
CD59/CD4, CD59/CD8, CD59/CD19, CD59/CD7/CD3, and CD16/CD7/CD3). The
cell/antibody combinations were mixed, incubated at room temperature for 30 minutes, and then centrifuged at 400g for 15 seconds to pellet the cells. After removal of excess antibody and washing twice
with 150-µL volumes of FACSFlow/BSA, the cells were resuspended to
300 µL in FACSFlow before flow cytometry studies. Cells were then
analyzed by flow cytometry using a FACScan flow cytometer and Lysis II
software. A minimum of 10,000 gated lymphocytes were collected on the
basis of low side scatter characteristics and either CD3, CD4, CD8, or
CD19 positivity.
Detection of the GPI-linked antigen CD48 expression by lymphocyte
subpopulations using whole blood lysis.
One hundred-microliter volumes of whole blood were stained as
described above with the following combinations of monoclonal antibodies (PE/Cy5 or FITC/PE/Cy5): CD48/CD3, CD48/CD4, CD48/CD8, CD48/CD19, CD48/CD7/CD3, and CD16/CD7/CD3. The cells were analyzed by
flow cytometry using a FACScan flow cytometer and Lysis II software. A
minimum of 5,000 gated events were collected on specific lymphocyte
populations identified by low side scatter characteristics and either
CD3, CD4, CD8, or CD19 positivity (summarized in
Fig 1). Data were stored as list mode files
for subsequent analysis.
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| Fig 1.
Summary of flow cytometry gating procedures for the study
of GPI-linked antigens (CD48 or CD59) by peripheral blood T cells, B
cells, and NK cells. Plot (A) shows a region (R1) drawn around
CD3+ cells with low side scatter characteristics; plot
(B) shows a region (R1) drawn around CD19+ cells with low
side scatter characteristics; plots (C and D) show the gating procedure
for NK cells. An initial region or anchor gate (R2) is set around
CD7+CD3 cells. This region is then applied
to an FSC versus SSC plot and a second region (R3) drawn around the
lymphocytes with low FSC and SSC characteristics. Only events (cells)
meeting these two criteria are collected and analyzed.
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Detection of GPI antigen expression by NK cells.
NK cells were identified by a multiple gating procedure using combined
fluorescence and light scattering characteristics (Fig 1). An initial
gate (R1) was drawn around CD7+CD3 cells
on a FL2 versus FL3 plot. This region was then applied to a FSC/SSC
plot and a second gate (R2) drawn around the lymphocyte population (low
FSC and SSC characteristics) to exclude contaminating granulocytes
(high FSC/SSC). A minimum of 5,000 events were acquired that met these
combined gating criteria. An FL1 histogram of CD48 expression (whole
blood lysis) or CD59 (separated cells) was then examined to determine
GPI-linked expression by NK cells. Purity of gating was assessed using
a CD16/CD7/CD3 combination of monoclonal antibodies and the same gating
criteria. In all cases CD16 positivity (a pan NK cell marker) exceeded
90%.
The sources and specificities of all monoclonal antibodies used for
immunophenotyping are summarized in Table 1.
| |
RESULTS |
The clinical, hematological, and laboratory features of the 19 patients
examined in the study are summarized in
Table 2. Nine patients had hypoplastic PNH,
eight hemolytic PNH, and a single patient, who originally presented
with hemolytic PNH, was nonhemolytic with only 0.5%
GPI RBCs and a normal hemoglobin. A single patient
showed spontaneous remission of PNH 15 years ago. Blood count
parameters showed a neutropenia (<2.0 × 109/L) in 9 of 19 patients, anemia in 13 of 19 patients, and thrombocytopenia in 8 of 19 patients (<150 × 109/L). A
lymphopenia was shown in 12 of 19 patients (<1.5 × 109/L). With the exception of case PNH030, GPI-deficient
cells were found in both the RBCs and neutrophils in all remaining
cases. Flow cytometry studies showed that in the majority of these
patients (16 of 18) the granulocyte PNH clone was larger than the RBC
PNH clone. However, there was wide variation in size of granulocyte clone, ranging from 1.4% to 99.5%. Similar wide variation (0.5% to
79.5%) was evident for the RBC PNH clone, further complicated by the
presence of partially deficient (type II) RBCs in seven patients.
Lymphocyte subset analysis and absolute numbers.
The absolute numbers of CD3+ (T cells)
CD3+CD4+ (T-helper),
CD3+CD8+ (T-cytotoxic) CD19+ (B
cells), and CD3CD16+ NK cells were
determined for each patient. The results
(Table 3)
showed a wide variety of abnormalities with no single patient having a
normal distribution of lymphocyte subsets. Absolute numbers of T cells
were generally normal, though three patients did show a T lymphopenia.
A mild CD4 lymphopenia was a common finding (10 of 19), with a CD8
lymphopenia in 5 of 19 and a mild CD8 lymphocytosis in two cases.
Absolute numbers of NK cells were markedly reduced in 17 of 19 (89%)
of patients. B-cell numbers were low in 11 of 19 cases (58%) and
borderline low in an additional 5 (26%) patients. A significant
increase in activated T cells (>20% expression of HLA-DR) was shown
in three patients.
Expression of GPI-linked proteins by T lymphocytes in patients with
PNH.
Of the 19 patients studied, GPI-deficient populations of T cells were
found in 15. The percentage of cells affected ranged from 0.2% to
21.6% (Fig 2 and Table 4). One case showed
a population of partial GPI-deficient cells comprising 8.1% of total T
cells. Further studies of CD4+ and CD8+ T-cell
components for individual patients revealed similar proportions of PNH
clones, and statistical analysis (paired t-test) showed no
significant difference (t = 2.48; degrees of freedom
[df] = 12; P = .028). Representative histograms of CD48 and
CD59 staining by T lymphocytes from PNH patients and normal controls
are shown in Fig 3. No
GPI lymphocytes were found in any of the normal
controls studied.
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| Fig 2.
Distribution of GPI (PNH) clones amongst T,
B, and NK lymphocytes for individual patients studied.
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| Fig 3.
(A) Representative fluorescence histogram profiles of the
expression of the GPI-anchored membrane protein CD48 (solid peaks) by
normal peripheral blood T cells, B cells, and NK cells. Negative
control antibody staining is shown by the clear histogram peaks.
No GPI-deficient cells were detected amongst normal lymphocytes. (B)
Representative staining profiles of CD48 expression by T cells, B
cells, and NK cells from three patients with PNH. Individual
histograms show clear bimodal distributions, with GPI-deficient and
GPI+ populations readily discernible in all cases. Two
particular features are noteworthy: (1) The lymphocytes from case
PNH063 show a partial GPI-deficiency. This is similar to their
partially deficient (type II) RBCs. (2) In two of the three patients
the proportion of GPI-deficent NK cells is much higher than the
proportions of B and T cells. (C) For comparison, typical CD59
histogram profiles of lymphocyte subpopulations for case PNH072 are
shown. Again, the proportion of NK cells that are GPI-deficient exceeds
that of B and T cells.
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Expression of GPI-linked proteins by B lymphocytes in patients with
PNH.
Studies of B lymphocyte populations identified by flow cytometry
characteristics of low SSC, and CD19 positivity revealed GPI populations in 16 patients, with 2 showing
partially deficient populations. The percentage of GPI-deficient cells
ranged from 0.8% to as high as 58.9% (Fig 2 and Table 4). No
GPI-deficient B cells were found in the normal controls studied.
Representative patient and control histograms of CD48 and CD59
expression are shown in Fig 3.
Expression of GPI-linked proteins by NK cells.
Using a novel gating strategy to identify NK cells,
GPI populations were found in 16 of the 17 cases
studied. The results ranged from 0.1% to 95% GPI-deficient cells (Fig
2 and Table 4). The two patients that also showed partially
GPI-deficient B-cell and/or T-cell populations also showed
similar partially deficient patterns of expression for NK cells. No
GPI populations were found amongst normal donor NK
cells studied by the same methods. Representative patient and control
histograms of CD48 and CD59 expression are shown in Fig 3. For
individual patients the percentage of GPI-deficient NK cells was
generally much higher than the percentage of GPI-deficient T cells and
B cells (Fig 2 and Table 4).
| |
DISCUSSION |
The investigation of peripheral blood lymphocyte subsets by flow
cytometry provides important information regarding the status of the
peripheral cellular immune system. What it does not provide is any
information regarding kinetics of cell production and destruction. For
normal healthy individuals, absolute numbers of lymphocyte subpopulations and levels of activation antigen expression fall within
tightly defined references ranges, and values outside these are found
in a range of disorders including infection, inflammatory conditions,
auto-immune disease, and malignancy. Results from this study clearly
show that lymphocyte subset abnormalities are a common feature of
patients with PNH. Low absolute numbers of B cells and NK cells were
the most frequent abnormalities detected (95% of patients), along with
a mild reduction in CD4+ T cells in 53% of patients. There
is little published data on lymphocyte subsets in PNH, though low
levels of circulating B cells and NK cells have been described
previously.25,26 These specific lymphocyte subset
abnormalities are not exclusive to PNH because similar findings have
been reported in patients with aplastic anemia.27-29 There
is no clear explanation for these abnormalities, though they may simply
reflect the general reduction in normal hematopoietic activity
associated with PNH. However, they highlight that cells of both innate
(NK cell) and specific (T and B cell) immune systems show quantitative
abnormalities. Whether these reductions are accompanied by any
functional impairment, clinical evidence of cellular or humoral
immunodeficiency, and subsequent increased susceptibility to infection
is unclear. However, none of the patients has suffered an excessive
number or severity of infections.
The second part of this study examined whether the PNH abnormality was
detectable in peripheral blood lymphocytes. Although previous studies
have unequivocally shown GPI-deficient B and T cells, the extent of
involvement of the NK cell lineage in vivo is less
certain.25,30 By applying a more precise gating strategy to
identify NK cells than has been previously used, we have clearly shown
the presence of GPI-deficient NK cells, and more significantly that
they comprise a significant proportion of total NK cells. Similarly, a
novel and precise multiparameter gating strategy was used to examine
expression of two GPI-linked antigens (CD48 and CD59) on peripheral
blood B cells, T cells, and T-cell subpopulations. Results showed that
in a high proportion of PNH patients all lymphoid cells comprised a
chimera of normal and PNH clones. The proportion of GPI-deficient cells
within individual lymphocyte lineages was highly variable, though in
81% of patients investigated with active disease, the percentage of
GPI-deficient NK cells was considerably higher than the percentage of
GPI-deficient B cells and T cells. The remaining cases showed either NK
cells alone affected or higher proportions of GPI-deficient T and B
cells than NK cells. The patient who had undergone spontaneous
remission of PNH had no detectable granulocyte or RBC PNH clones, but
significant proportions of GPI-deficient T and B cells were detected.
In addition, partially deficient lymphocytes were detected in only two
of the seven patients with type II (partially deficient) RBCs.
To offer a potential explanation for these findings it is firstly
necessary to review current concepts of lymphopoiesis and contributory
factors affecting development and production of lymphocytes. The
evidence for a common lymphoid progenitor cell is very strong, as mice
deficient for the stem cell transcription factor Ikaros produce
cells of the myeloid lineage but no T, B, or NK cells.31 Cell-culture studies suggest that B progenitors diverge from the developmental pathway earlier than T and NK cells, further
differentiation of the lymphoid precursor cell produces
a bipotential T/NK precursor.32 At this point, commitment
to T-cell or NK-cell lineage either requires migration to the thymus or
remaining in the bone marrow, respectively, though a murine thymic
progenitor cell can generate T, B, and NK cells (reviewed in detail by
Spits et al).33 More recent studies in mice have identified
a common lymphoid progenitor cell in the bone marrow that has no
self-renewal capacity but can generate T, B, and NK
cells.34 A critical component of B and T lymphoid
development is generation of antigen receptor diversity together with
removal of potentially harmful autoreactive cells created by this
process. For B cells the removal of autoreactive cells occurs in bone
marrow, and up to 90% of newly produced IgM+ B cells
survive for only a short period of time. Similarly, there is
considerable intrathymic T-cell death with only small numbers of naive
T cells surviving the selection process. NK precursors are thought to
differentiate in the bone marrow relatively unhindered into mature NK
cells and develop their limited receptor diversity by an alternative,
less understood mechanism.35 Therefore, for individual PNH
patients in whom the majority of hematopoiesis is derived from stem
cells with a PIG-A mutation, the probability of a PNH lymphoid
progenitor producing a mature, naive GPI-deficient B cell or T cell is
inherently low due to high levels of ineffective lymphopoiesis and
negative selection processes. For NK cells the opposite is true, as
their precursors and differentiation pathways are not subject to the
same levels of scrutiny as B and T cells. However, if a naive
GPI-deficient B or T cell survives the differentiation and selection
process, it will have the capacity to remain in the peripheral lymphoid
system for many years. This latter point in particular is supported by
the results from patient PNH030 and other reports of patients in
long-term clinical remission for PNH with GPI-deficient T-and
B-lymphocyte populations but no detectable granulocyte or RBC
clones.21,22
As many patients often have prolonged periods of illness before the
onset of the characteristic pathology of PNH, it is not possible to
predict the exact time point at which the PIG-A mutation occurs or when
the progeny of the GPI-deficient stem cell begins to become detectable.
We are currently undertaking serial studies on patients to develop a
model of cell kinetics in PNH and to analyze whether these can be used
to predict the clinical course of the disease. We propose that when PNH
stem cells become the predominant source of hematopoietic elements, the
myeloid/erythroid series is always the first to be affected, and this
leads to the characteristic pathology of severe hemolytic anemia
and/or aplasia. Then, depending on the degree of lymphopoietic
activity, GPI-deficient lymphocytes emerge, beginning with NK cells and
followed eventually by B and T cells. Similarly, if recovery from PNH
occurs with restoration of hematopoiesis, the myeloid series will be
the first to recover, followed by NK cells. If GPI-deficient T- and
B-cell populations were present, then because of their long life span, they would remain detectable for many years. However what is not known
is what effect immunosuppressive therapy and/or deficiency of
GPI antigens from the lymphocyte membrane has on lymphopoiesis.
To test this model, we are undertaking long-term studies of these
patients and also studying cases in which clinical remission of PNH has
occurred. It may be that sequential studies of these patients, and
particularly the composition of lymphocytes, may provide a better
understanding of the natural history and kinetics of the disease. An
additional dividend is the probability that the results will also
provide important insights into in vivo adult lymphopoiesis.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted May 4, 1998.
Address reprint request to Stephen J. Richards, PhD,
Haematological Malignancy Diagnostic Service, The Algernon Firth
Building, Leeds General Infirmary, Leeds, LS1 3EX, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge the support of Cymbus Biosciences
for generous donation of monoclonal antibodies used in this study. We
also thank Drs J. Yin, R.G. Hughes, M. Layton, M. Laffan, L.A. Parapia,
M. Hill, D. Bareford, and M. Hamilton for providing patient samples.
| |
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Abstract
Introduction
Abstract