Blood, 15 July 2001, Vol. 98, No. 2, pp. 489-491
BRIEF REPORT
Factors influencing B lymphopoiesis after allogeneic
hematopoietic cell transplantation
Jan Storek,
Denise Wells,
Monja A. Dawson,
Barry Storer, and
David G. Maloney
From the Fred Hutchinson Cancer Research Center,
University of Washington, and Hematologics, Seattle, WA.
 |
Abstract |
In 93 allograft recipients, the numbers of marrow B-cell precursors
on days 80 and 365 correlated with the counts of circulating B cells,
suggesting that the posttransplantation B-cell deficiency is at least
in part due to insufficient B lymphopoiesis. Factors that could affect
B lymphopoiesis were evaluated. The number of marrow B-cell
precursors on days 30 and 80 was at least 4-fold lower in patients with
grade 2 to 4 acute graft-versus-host disease (GVHD) compared with
patients with grade 0 to 1 acute GVHD. The number of B-cell precursors
on day 365 was 18-fold lower in patients with extensive chronic GVHD
compared with patients with no or limited chronic GVHD. The number of
B-cell precursors was not related to CD34 cell dose, type of transplant
(marrow versus blood stem cells), donor age, or patient age. It was
concluded that posttransplantation B-cell deficiency results in part
from inhibition of B lymphopoiesis by GVHD and/or its treatment.
(Blood. 2001;98:489-491)
© 2001 by The American Society of Hematology.
| |
Introduction |
Allogeneic hematopoietic cell transplant (HCT)
recipients are immunocompromised for more than 1 year after
grafting.1,2 Quantitative B-cell deficiency plays a role
in the susceptibility of transplant recipients to
infections.3 The quantitative B-cell deficiency may be due
in part to low B-cell production (B lymphopoiesis) in the marrow of HCT
recipients. Quantitative deficiency of B-cell precursors is observed in
virtually all patients in the first month and in some patients for more
than 1 year after grafting.4-6 We set out to determine
which factors might influence B-cell-precursor quantity (index of B
lymphopoiesis) in the marrow of HCT recipients. We hypothesized that
the following factors might play a role: (1) CD34 cell dose; (2) graft
type (marrow versus blood stem cells), as blood stem cell recipients
receive 3 times more CD34 cells and 18 times fewer B-cell precursors
compared with marrow recipients7; (3) patient or donor
age, as young, normal individuals have more B-cell precursors than
older individuals8-10; and (4) acute or chronic
graft-versus-host disease (GVHD) or its treatment, as chronic GVHD
and/or its treatment were associated with low marrow content of B-cell
precursors in a small clinical study6 and B-lymphopoiesis
was inhibited by glucocorticoids in a murine
study.11,12
| |
Study design |
Since one of the goals of the study was to determine how B
lymphopoiesis was influenced by the graft type, we studied patients randomized to receive either marrow or filgrastim-mobilized blood stem
cells.13 Of 135 patients treated randomly with either
transplant type at the Fred Hutchinson Cancer Research Center, 123 patients agreed to institutional review board-approved studies
of immune reconstitution. Twelve patients died by day 30. One patient
received marrow instead of blood stem cells and was excluded from
analysis. For 17 patients, no marrow samples were submitted for the
quantitation of B-cell precursors. Thus, 93 patients were evaluable for
the quantitation of B-cell precursors between 1 and 12 months after transplantation. Their characteristics, including data on CD34 cell
dose, graft type, age, and acute and chronic GVHD, are displayed in
Table 1.
Marrow was aspirated on approximately days 30, 80, and 365 after
transplantation. Cells were stained with CD19 antibody conjugated to
phycoerythrin; CD45 antibody conjugated to peridinin-chlorophyll; and
CD2, CD3, CD5, and CD33 antibodies conjugated to fluorescein isothiocyanate (Becton Dickinson, San Jose, CA). Erythrocytes were
lysed by NH4Cl (0.83%, buffered with KHCO3, pH
7.2). Flow cytometry data acquisition was performed on CytoronAbsolute
(Ortho, Raritan, NJ) or FACSCalibur (Becton Dickinson) instruments.
Data analysis was performed by means of Winlist software (Verity House, Topsham, ME). Cellular debris and nonviable cells were first eliminated by forward- versus side-scatter gating. Then, the percentage of B-cell
precursors
(CD19+CD45lowCD2
/CD3/CD5/CD33)
of total CD45+ cells was determined.9,14,15 We
used the percentage of B-cell precursors among marrow CD45+
cells corrected for marrow cellularity (multiplied by the percentage of
normal cellularity divided by 100) and for the ratio of myeloid and
lymphoid cells to erythrocyte precursors (M:E ratio) since erythrocyte
precursors do not express CD45 (multiplied by M:E ratio divided by
[1 + M:E ratio]). For example, if 10% of marrow CD45+ cells were B-cell precursors, the hematopoietic
cell-to-fat ratio was 25:75 (50% normal), and the M:E ratio was 4:1,
then the corrected percentage of B-cell precursors was
10 × 0.5 × 0.8 = 4. Marrow cellularity and M:E ratio were
estimated from standard decalcified biopsy sections or aspirated
particle sections stained with hematoxylin and eosin. Marrow specimens
from patients whose hematologic malignancy relapsed were not evaluated
unless relapse was determined only by a polymerase chain reaction.
| |
Results and discussion |
Patients who developed grade 2 to 4 acute GVHD by day 30 had a
lower percentage of B-cell precursors on day 30 compared with patients
who developed grade 0 to 1 acute GVHD by day 30 (median, 0.1 versus
0.4; P = .03, Mann-Whitney rank-sum test). Patients who
developed grade 2 to 4 acute GVHD by day 90 had a lower percentage of
B-cell precursors on day 80 compared with patients who developed grade
0 to 1 acute GVHD by day 90 (median, 0 versus 0.9;
P < .001). Patients who developed clinical extensive,
chronic GVHD in the first posttransplantation year had a lower
percentage of B-cell precursors on day 365 compared with patients who
developed no or limited chronic GVHD in the first year (median, 0.1 versus 1.8; P < .001) (Figure
1, upper panels). We could not separate the effect of acute or chronic GVHD from the effect of glucocorticoids as there were few patients on glucocorticoids without GVHD and few
patients off glucocorticoids with GVHD. The GVHD and/or its treatment
depressed both early (CD19low) and late
(CD19high) B-cell precursors; however, the early precursors
appeared depressed to a greater degree (Figure 1, middle and
lower panels).
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| Figure 1.
Percentage of B-cell precursors among CD45+
marrow cells (corrected for marrow cellularity and for M:E ratio).
Total, CD19low (early), and CD19high (late)
B-cell precursors were quantitated (upper, middle, and lower panels,
respectively). The corrected percentage of total B-cell precursors on
day 30 was lower in patients who developed grade 2 to 4 acute GVHD by
day 30 (n = 19) than in patients who did not do so (n = 52). The
corrected percentage of total B-cell precursors on day 80 was lower in
patients who developed grade 2 to 4 acute GVHD by day 90 (n = 54)
than in patients who did not do so (n = 20). The corrected percentage
of total B-cell precursors on day 365 was lower in patients who
developed clinical extensive, chronic GVHD (n = 28) than in patients
who developed no or limited chronic GVHD (n = 23) in the first
posttransplantation year. The corrected percentages of the early
precursors appear to be influenced by the GVHD and/or its treatment to
a greater extent than the late precursors. The squares denote medians
and the vertical bars denote 25th to 75th percentiles. In all 9 graphs,
the difference between the 2 groups is significant
(P < .05 for day 30; P < .01 for day 80;
P < .001 for day 365).
|
|
Peripheral blood stem cell recipients and marrow recipients did not
significantly differ in the percentage of total B-cell precursors on
day 30, 80, or 365. Also, there was no significant correlation between
CD34 cell dose, patient age, or donor age and the percentage of B-cell
precursors on either day 30 or day 80 (Spearman rank correlation test).
Similarly, there was no significant correlation between CD34 cell dose
or patient age and the percentage of B-cell precursors on day 365. However, there was an inverse correlation between the percentage of
B-cell precursors on day 365 and donor age
(r = 
0.32; P = .02). In
multivariate analysis, the percentage of B-cell precursors on day 365 was significantly associated with clinical extensive chronic GVHD
(P < .001) and not with donor age (linear regression
analysis of log-transformed percentages).
We also evaluated whether a low percentage of B-cell precursors was
associated with low circulating B-cell counts (determined as described
[mononuclear cells expressing CD19 or CD20 and not brightly expressing
CD3, CD13, CD14, CD16, CD56, CD10, or CD34]7 on days 30, 80, 180, and 365 after transplantation). The following correlations
were found: (1) the percentage of B-cell precursors on day 80 and
B-cell counts on day 80 (Spearman correlation coefficient r = 0.25; P = .057); (2) the percentage of
B-cell precursors on day 80 and B-cell counts on day 180 (r = 0.35; P = .031); and (3) the percentage
of B-cell precursors on day 365 and B-cell counts on day 365 (r = 0.50; P = .002).
The results show that between days 80 and 365 after transplantation,
the size of the circulating B-cell pool depends on B lymphopoiesis
and that GVHD and/or its treatment is the major factor influencing B
lymphopoiesis. CD34 cell dose, graft type (blood stem cells versus
marrow), patient age, and donor age do not affect B lymphopoiesis significantly.
In the first 3 months after grafting, blood stem cell recipients have
significantly higher counts of circulating B cells than marrow
recipients.7,16 Because the percentage of B-cell
precursors on days 30 and 80 was similar in blood stem cell and marrow
recipients, the higher circulating B-cell count in blood stem cell
recipients is most likely due to the higher content of mature B cells
in blood stem cell grafts compared with marrow
grafts,7,16,17 rather than to increased B lymphopoiesis.
There are several possible mechanisms by which patients with GVHD have
decreased B lymphopoiesis. The first is through production of cytokines
from activated cells that may inhibit B lymphopoiesis. Examples include interferon-
produced by T cells18 or
interleukin-1 produced by macrophages during the GVHD-associated
"cytokine storm."12,19,20 The second possible
mechanism is the destruction of marrow stromal cells, which support B
lymphopoiesis,21 by donor lymphocytes (graft-versus-stroma
reaction).22,23 The third mechanism is suppression of
B lymphopoiesis through treatments given for GVHD, eg,
glucocorticoids.11,12 Although some of these mechanisms might be amenable to a therapeutic intervention, significant
improvement of B lymphopoiesis is likely to be achieved only through
effective GVHD prophylaxis.
| |
Footnotes |
Submitted December 6, 2000; accepted March 22, 2001.
Supported by National Institutes of Health grants CA68496, AI46108,
CA18221, and CA18029.
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: Jan Storek, FHCRC, D1-100, 1100 Fairview Ave N,
Seattle, WA 98109-1024; e-mail: jstorek{at}fhcrc.org.
| |
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Abstract
Introduction
