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IMMUNOBIOLOGY
From the Haematological Malignancy Diagnostic Service,
Leeds General Infirmary, Leeds, United Kingdom.
Peripheral blood B cells in patients with paroxysmal nocturnal
hemoglobinuria (PNH) comprise variable mixtures of normal B cells
produced before the onset of disease and glycosylphosphatidylinositol (GPI)-deficient B cells derived from the PNH hematopoietic stem cell.
In a detailed phenotypic analysis of 29 patients with PNH, this study
shows consistent phenotypic differences between PNH B cells and
residual normal B cells. In the majority of patients with active
disease, PNH B cells comprised mainly naive cells with a
CD27 Recent studies have shown that coexpression of the
tumor necrosis factor receptor superfamily member CD27 and the
receptor-type protein tyrosine phosphatase CD148 by human peripheral
blood B cells defines the memory B-cell compartment generated by
germinal center reaction.1-3 These
CD19+CD27+ cells show somatically mutated
immunoglobulin (Ig) variable gene regions and can be subdivided into 2 phenotypically distinct populations of memory B cells: (1) Ig
class-switched cells expressing surface IgG or IgA and (2) IgM only and
IgM/IgD-expressing memory cells.2-4 Further evidence that
CD27 is a marker for memory B cells is shown by the findings that cord
blood B cells are predominantly CD27 Paroxysmal nocturnal hemoglobinuria (PNH) is a unique condition
in which somatic mutation of the X-linked PIG-A gene within a hematopoietic stem cell results in a partial or absolute deficiency of glycosylphosphatidylinositol (GPI)-linked membrane
proteins.8-13 Following onset of the disorder and failure
of normal hematopoiesis, new blood cell formation in patients with
large PNH clones is almost entirely derived from the PNH stem cell.
Nevertheless, in the majority of patients, residual populations of
normal myeloid and erythroid cells produced by the few remaining normal
stem cells can be detected. The majority of T and B lymphocytes are usually normal with only relatively small PNH populations present even
in patients with over 90% of their hematopoiesis derived from the PNH
clone. This is due to the fact that many of these are long-lived cells
produced before the onset of PNH.
We have shown previously that PNH can be used as a model with which to
study the immune system and that the absence of GPI-linked antigens can
be used as a biologic marker with which to examine production of cells
following the onset of PNH.14 We have used this approach
to study peripheral blood B cells to examine the definition of naive
and memory B cells, the phenotypic characteristics of the memory B-cell
compartment in the peripheral blood, and the effect of aging on B-cell
production. A more detailed understanding of peripheral memory B-cell
compartment and the humoral immune response is important for a number
of reasons including monitoring B-cell recovery after bone marrow
transplantation or after chemotherapy and potentially monitoring the
effectiveness of vaccination.
Using sensitive multicolor flow cytometry techniques, GPI-deficient
(GPI Patients
Isolation of leukocytes by differential erythrolysis with
ammonium chloride
Determination of the absolute numbers of B lymphocytes An initial whole blood lysis screen was performed to assess the proportions and absolute numbers of B cells using a prestandardized combination of directly conjugated CD3/HLA-DR/CD19 monoclonal antibodies (fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE:Cy5) and an established protocol.16 The cells were analyzed using a FACScan flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson) collecting a minimum of 1 × 104 gated lymphocyte events identified by low forward scatter (FSC) and low side scatter (SSC) characteristics. Absolute numbers of B cells were calculated using the Sysmex lymphocyte count multiplied by the percentage of lymphocytes expressing CD19 divided by 100.Analysis of CD27 expression on normal and paroxysmal nocturnal hemoglobinuria B cells Volumes (100 µL) of whole blood were stained as for absolute B-cell counts with 30-µL volumes of the following combination of fluorochrome-conjugated monoclonal antibodies: CD48Fitc/CD27PE/CD19PE:Cy5. Cells were analyzed by flow cytometry using a FACScan flow cytometer with CellQuest software. A minimum of 1 × 103 gated events were collected on B-lymphocyte populations identified by low SSC characteristics and CD19 positivity. Data were stored as list mode files for subsequent analysis. Quadrant statistical analyses of dot-plots of CD48 versus either CD27 were undertaken, and the proportions of normal and PNH lymphocytes expressing CD27 calculated. Absolute numbers of GPI naive
B cells were calculated by multiplying the absolute numbers of B cells
by the percentage of CD48CD27 cells,
divided by 100.
Analysis of Ig heavy-chain expression on normal and paroxysmal nocturnal hemoglobinuria B cells Immunoglobulin heavy-chain expression (IgM and IgG) was examined on normal and GPI B-cell subsets of 10 patients using
3-color flow cytometry. Leukocytes (1 × 106) were
stained in microtiter plate wells with 30 µL of the following combinations of fluorochrome-conjugated monoclonal antibodies: (Fitc/PE/PE:Cy5) IgM/CD48/CD19 and IgG/CD48/CD19. 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
500 µL in FACSFlow prior to flow cytometry studies and analyzed by
flow cytometry. A minimum of 1 × 104 gated B cells was
collected on the basis of low SSC characteristics and CD19 positivity.
The proportions of normal and PNH B cells expressing IgM or IgG were
derived from quadrant statistical analysis of CD48 versus Ig
heavy-chain dot-plots. In addition, analysis of CD48/IgD/CD19
expression was undertaken in 4 cases.
Monoclonal antibodies The sources and specificities of all monoclonal antibodies used for immunophenotyping were as follows: CD3 (OKT3 Fitc) and HLA-DR (L243 PE) from American Type Culture Collection (ATCC); CD48 (MEM102 Fitc and PE) from Cymbus Biotechnology Limited, Chandler's Ford, Hampshire, UK; CD19 (FMC63 PE:Cy5) from Professor Zola, Flinders Medical Centre, Australia; and CD27 (M-T271 PE), anti-IgD (IA6-2 PE), anti-IgG (G18-145 Fitc), anti-IgM (G20-127 Fitc) from Pharmingen, San Diego, CA.
The clinical and laboratory features of the 29 patients are
presented in Table 1. Fifteen patients
had hemolytic PNH, 11 had hypoplastic disease, and 2 patients presented
with thrombotic disease. Two of the hemolytic patients also had
concurrent thrombotic disease, and 2 patients had morphologic evidence
of myelodysplastic syndrome. Two patients (PNH104 and PNH008) no longer
had detectable PNH red cells or granulocyte clones in the peripheral
blood. Patient PNH104 originally presented in 1962 with severe
hemolytic PNH; this persisted for a number of years with attacks of
macroscopic hemoglobinuria ceasing in 1976. He currently has
myelodysplastic syndrome. Expression of GPI-linked antigens on red
cells and granulocytes was abnormal, although the flow cytometry
profiles of CD55 and CD59 expression were not those seen in classical
PNH. Patient 008 originally presented with hemolytic PNH 12 years ago
and is currently in clinical remission. She has no detectable
granulocyte or red cell PNH clones by flow cytometry in the
peripheral blood.
The median size of the granulocyte clone for the 27 patients with active PNH was 77.0%. This was considered as a reliable indicator that the majority of hematopoiesis was clonally derived from the PNH stem cell. Absolute B-cell counts were low (< 0.06 × 109/L) in 15 of 29 patients (52%) and borderline low (> 0.06 × 109/L but < 0.1 × 109/L) in an additional 7 patients. The median B-cell count was 0.06 × 109/L (observed range 0.01-0.33 × 109/L). Detection of paroxysmal nocturnal hemoglobinuria B-cell clones and relationship with the granulocyte paroxysmal nocturnal hemoglobinuria clone size Of the 29 patients studied, PNH B cells were detected in all cases including the 2 patients with spontaneous remission. The percentage of GPI B cells showed a wide variation (0.14%-54.4%,
median 3.05%) though in 86% of cases the clone comprised less than
10% of total B cells. To determine whether this variability was
related to the activity of the PNH stem cell, correlations between the
levels of PNH B cells and PNH granulocytes were examined. No
significant correlations were found between the proportions (Spearman
[rs] 0.131; P = .45) or absolute numbers
(Spearman (rs) 0.16; P = .42) of PNH B cells and PNH granulocytes.
Relationships between age and absolute numbers of paroxysmal nocturnal hemoglobinuria B cells One explanation for the wide variation in the proportion of PNH B cells is that patient age may affect the ability to produce B cells. Because PNH B cells are unequivocally derived from a bone marrow hematopoietic stem cell, we examined the relationship between absolute numbers of PNH B cells versus patient age as a measure of B-cell production. No significant correlation was found (Spearman [rs] 0.017; P = .93). In a more specific analysis of the 19 patients where the majority of hematopoiesis was derived from the PNH stem cell (ie, a > 50% PNH granulocyte clone) again no significant correlation was found (Spearman [rs] 0.062; P = .80). These findings show that the proportion of PNH B cells present in any individual patient is not directly related to the size of the granulocyte PNH clone nor to patient age. Furthermore, these studies confirm that new B cells are continually produced throughout adult life and that the levels of production appear not to decline with age (Figure 1).
CD27 expression by CD48+
and CD48 ) B cells with residual normal B cells
(CD48+) in the same patient (Table
2) showed absent or reduced levels of
CD27 expression within the PNH component. These differences were
statistically significant (paired t test: t =  9.96;
degrees of freedom [df], 28; P < .0001). A series of
representative flow cytometry dot-plots of CD48 versus CD27 for
CD19+ gated B cells are shown in Figure
2 and illustrate differential CD27
expression between GPI B cells and normal B cells. The
GPI B cells detected in patients with a recent onset of
PNH were predominantly of a naive (CD27) phenotype
consistent with recent production. The coexisting GPI+ B
cells in these patients showed normal proportions of CD27+
cells. In marked contrast to this, the residual normal B-cell components in patients with large PNH granulocyte clones and a long
history of PNH were predominantly of a memory (CD27+)
phenotype. There are 2 possible mechanisms that contribute to this: (1)
the long-term failure in production of new naive GPI+ B
cells and (2) conversion of the existing normal CD27 B
cells, produced before the onset of PNH, to a memory phenotype. The
highest proportions of CD27+ PNH B cells were found in
patients with long duration of disease and in the 2 patients in whom
spontaneous remission of PNH had occurred.
Previous studies have established that CD27 expression on normal
peripheral blood B cells increases with age.5 To determine whether CD27 expression on PNH B cells was related to disease duration,
we examined the correlation between percentage CD27 expression by the
PNH B cells and time from the date of onset of clinical symptoms of
PNH. Statistical analysis (Figure 3), not
unexpectedly, revealed a significant correlation (Spearman [rs] 0.403; P = .030) between disease
duration and the proportion of CD27+ PNH B cells (ie,
memory PNH B cells).
Surface IgM and IgG expression by
normal (CD48+) and paroxysmal nocturnal
hemoglobinuria (CD48 B cells.
IgM and IgG expression was studied in 10 patients, 8 with active
disease and 2 in remission, using 3-color flow cytometry. This analysis
(Table 3 and Figure
4) showed that the PNH B-cell component
in patients with active disease (hemolytic or hypoplastic) were
predominantly IgM+IgG(CD27),
that is, a naive phenotype consistent with recent production. Furthermore, analysis of membrane IgD expression in 3 patients (PNH008,
PNH042, and PNH069) showed that the strength of expression of IgD was
considerably higher on PNH B cells compared to residual normal B cells
(Figure 4). The PNH B cells in patient PNH104, who developed PNH 36 years ago, showed a high proportion of memory cells with significant
populations of non-class-switched (IgM+) and
class-switched (IgG+) cells.
The residual normal B-cell component in patients where the majority of
hematopoiesis was derived from the PNH stem cell (ie, > 90% of
granulocytes GPI
Analysis of CD27 expression by normal and GPI Analysis of the normal (GPI+) B-cell component was
more complex, particularly in patients with only small PNH granulocyte
clones. In these patients hematopoiesis is variably maintained from
both normal and PNH stem cells, and the normal B cells generally
comprised normal proportions of CD27+ components. In cases
with a longer duration of disease and a large PNH granulocyte clone
(ie, hematopoiesis maintained largely from the PNH stem cells), the
residual normal B cells comprised mainly B cells generated before the
onset of PNH. The majority of these were CD27+ (memory
phenotype) with very few detectable CD27 A more detailed analysis of Ig heavy-chain isotype expression in 10 patients (8 with active PNH and 2 in remission) showed significant
phenotypic differences between PNH B cells and normal B cells. In 4 patients, all with a long duration of active disease and granulocyte
clones of more than 90%, the residual normal B-cell component was
predominantly CD27+, with the majority of cells expressing
IgM and only small proportions of class-switched (IgG+)
cells. These results demonstrate that the majority of circulating memory B cells in fact comprise non-class-switched cells. It is also
evident from these patients that in the absence of any new naive B-cell
formation, over a period of 3 to 5 years the majority of normal B cells
convert to a memory phenotype. Sequential studies of the residual
normal B-cell component in a cohort of patients with large PNH
granulocyte clones and a recent onset of disease should further clarify
this. In contrast to these findings for GPI+ B cells, the
PNH B-cell component in all patients with active PNH was predominantly
CD27 In the absence of a simple in vitro functional assay to test whether
these CD27 Using multicolor flow cytometry we were able to detect
GPI
We are indebted to the many clinicians throughout the United Kingdom for providing patient samples. The authors gratefully acknowledge the support of Cymbus Biosciences for generous donation of monoclonal antibodies used in this study.
Submitted April 26, 2000; accepted July 21, 2000.
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: Stephen J. Richards, Haematological Malignancy Diagnostic Service, The Algernon Firth Building, Leeds General Infirmary, Leeds, LS1 3EX, United Kingdom; e-mail: stephenr{at}pathology.leeds.ac.uk.
1.
Tangye SG, Liu YJ, Aversa G, Phillips JH, de Vries JE.
Identification of functional human splenic memory B cells by expression of CD148 and CD27.
J Exp Med.
1998;188:1691-1703 2. Klein U, Goossens T, Fischer M, et al. Somatic hypermutation in normal and transformed human B cells. Immunol Rev. 1998;162:261-280[Medline] [Order article via Infotrieve].
3.
Klein U, Jajewski K, Küppers R.
Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells.
J Exp Med.
1998;188:1679-1689
4.
Klein U, Küppers R, Jajewski K.
Evidence for a large compartment of IgM-expressing memory B cells in humans.
Blood.
1997;89:1288-1298 5. Agematsu K, Nagumo H, Yang FC, et al. B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production. Eur J Immunol. 1997;27:2073-2079[Medline] [Order article via Infotrieve]. 6. Huang C, Stewart AK, Schwartz RS, Stollar BD. Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes. J Clin Invest. 1992;89:1331-1343. 7. Klein U, Küppers R, Jajewski K. Human IgM+IgD+ B cells, the major B cell subset in the peripheral blood, express V kappa genes with no or little somatic mutation throughout life. Eur J Immunol. 1993;23:3272-3277[Medline] [Order article via Infotrieve]. 8. Takeda J, Miyata T, Kawagoe K, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993;73:703-711[Medline] [Order article via Infotrieve]. 9. Bessler M, Mason PJ, Hillmen P. Luzzatto L. Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria. Lancet. 1994;343:951-953[Medline] [Order article via Infotrieve]. 10. Bessler M, Mason PJ, Hillmen P. Luzzatto L. Mutations in the PIG-A gene causing partial deficiency of GPI-linked surface proteins (PNH II) in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1994;87:863-866[Medline] [Order article via Infotrieve]. 11. Bessler M, Mason PJ, Hillmen P, et al. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene. EMBO J. 1994;13:110-117[Medline] [Order article via Infotrieve].
12.
Miyata T, Yamada N, Iida Y, et al.
Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
1994;330:249-255
13.
Ware RE, Rosse WF, Howard TA.
Mutations within the Piga gene in patients with paroxysmal nocturnal hemoglobinuria.
Blood.
1994;83:2418-2422
14.
Richards SJ, Morgan GJ, Hillmen P.
Analysis of T cells in paroxysmal nocturnal haemoglobinuria (PNH) provides direct evidence that thymic T cell production declines with age.
Blood.
1999;94:2790-2799
15.
Hillmen P, Bessler M, Crawford DH, Luzzatto L.
Production and characterization of lymphoblastoid cell lines with the paroxysmal nocturnal hemoglobinuria phenotype.
Blood.
1993;81:193-199
16.
Richards SJ, Norfolk DR, Swirsky DM, Hillmen P.
Lymphocyte subset analysis and glycosylphosphatidylinositol phenotype in patients with paroxysmal nocturnal hemoglobinuria.
Blood.
1998;92:1799-1806 17. Tseng JE, Hall SE, Howard TA, Ware RE. Phenotypic and functional analysis of lymphocytes in paroxysmal nocturnal hemoglobinuria. Am J Hematol. 1995;50:244-253[Medline] [Order article via Infotrieve]. 18. Kwong YL, Lee CP, Chan TK, Chan LC. Flow cytometric measurement of glycosylphosphatidyl-inositol-linked surface proteins on blood cells of patients with paroxysmal nocturnal hemoglobinuria. Am J Clin Pathol. 1994;102:30-35[Medline] [Order article via Infotrieve]. 19. Navenot JM, Bernard D, Harousseau JL, Muller JY, Blanchard D. Expression of glycosyl-phosphatidylinositol-linked glycoproteins in blood cells from paroxysmal nocturnal haemoglobinuria patients. A flow cytometry study using CD55, CD58 and CD59 monoclonal antibodies. Leuk Lymphoma. 1996;21:143-151[Medline] [Order article via Infotrieve].
20.
Hall SE, Rosse WF.
The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria.
Blood.
1996;87:5332-5340 21. Jin JY, Tooze JA, Marsh JC, Gordon-Smith EC. Glycosylphosphatidyl-inositol (GPI)-linked protein deficiency on the platelets of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinuria: two distinct patterns correlating with expression on neutrophils. Br J Haematol. 1997;96:493-496[Medline] [Order article via Infotrieve]. 22. Townsend SE, Weintraub BC, Goodnow CC. Growing up on the streets: why B cell development differs from T-cell development. Immunol Today. 1999;20:217-220[Medline] [Order article via Infotrieve].
23.
Guillaume T, Rubinstein DB, Symann M.
Immune reconstitution and immunotherapy after autologous hematopoietic stem cell transplant.
Blood.
1998;92:1471-1490 24. Nunez C, Nishimoto N, Gartland GL, et al. B cells are generated throughout life in humans. J Immunol. 1996;156:866-872[Abstract].
© 2000 by The American Society of Hematology.
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  M. Seifert and R. Kuppers Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation J. Exp. Med., November 23, 2009; 206(12): 2659 - 2669. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
