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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-03-0896.
IMMUNOBIOLOGY
From the Immunobiology and Cancer Program, Oklahoma
Medical Research Foundation; Department of Medicine, University of
Oklahoma Health Sciences Center; and St Anthony Hospital, Oklahoma
City.
This study addressed several questions concerning age-related
changes in human B lymphopoiesis. The relative abundance of pro-B,
pre-B, immature, naive, and mature B cells among the CD19+
lymphocyte fraction of human bone marrow was found not to change appreciably over the interval between 24 and 88 years of age. Moreover,
proliferation of pro-B and large pre-B cells in adult marrow equaled
that observed with fetal marrow specimens. Exceptionally low numbers of
lymphocyte precursors were found in some marrow samples, and the values
obtained were used to determine parameters that best reflect B
lymphopoiesis. Cord blood always contained higher incidences of
functional precursors than adult cells. However, sorted
CD34+ Lin Humoral immune responses are known to be
compromised in the aged, and decreased numbers of B and T lymphocytes
parallel increases in natural killer (NK) cells.1-3 Animal
studies have revealed age-related changes in B-lymphocyte production
within bone marrow (BM).4-6 Numbers of pro-B cells that
can be resolved by flow cytometry are relatively stable with age, but
pre-B cells decline substantially.4,7 Incorporation of
bromodeoxyuridine (BrdU) into dividing cells indicates that
B-cell production is diminished in old mice.7 This
corresponds to the reduced entry of newly formed cells to the
long-lived lymphocyte pool.7,8 Functional precursor
activity may decline because of a diminished support capacity by marrow
stromal cells.4,9 All of these observations indicate that
peripheral B-lymphocyte populations in the aged may be replenished with
fewer cells from marrow, with potential consequences for the repertoire
of available antibody specificities. However, it is unclear if such
findings are completely applicable to humans.
Animal studies have also been informative about molecular mechanisms
and steps in B lymphopoiesis. Rare hematopoietic stem cells transit a
number of "compartments" that are defined on the basis of cell
surface markers. In reality, this represents a continuum where genes
are expressed and repressed in a temporal, but not necessarily
synchronous, fashion. Early precursors have multiple differentiation
options and may express transcription factors in a way that does not
reflect their ultimate fate.10,11 Cells with potential for
differentiation to lymphoid, but not myeloid or erythroid, cells have
been referred to as common lymphoid progenitors.12,13 These
Lin Knowledge of early B-lymphocyte progenitors in humans lags behind that
for mice, and there are important differences. Interleukin 7 (IL-7) is
essential to this process in mice but not in humans, and no combination
of cytokines has been identified that supports vigorous human
lymphocyte formation in culture.19-21 Indeed, the most
effective models for human B lymphopoiesis use murine stromal cells
that provide uncharacterized stimuli.22 Cytokines and hormones have been identified that selectively inhibit B-lymphocyte formation in mice.13,23-27 It is important to know if
lymphopoiesis is altered as humans age because such negative factors
increase. Some investigative techniques are not appropriate with
specimens of human marrow, and the yield of hematopoietic cells is
variable. Cellularity decreases with age in some but not all human
bones,28,29 and most studies have been performed on marrow
aspirates contaminated by peripheral blood.30,31
Despite these species differences and technical difficulties, progress
is being made in understanding the process in humans. For example,
evidence indicates that CD10+CD19 Expression of Rag genes in marrow of mature individuals might
suggest that immunoglobulin genes are being
rearranged.34,35 Unfortunately, little information has
been available about proliferation36,37 or differentiation
activity in humans, and it is possible this slows with age. Thus,
although the incidence of lymphocytes has been known to decline between
birth and puberty, there was an insufficient understanding of changes
that may occur subsequently.31,35,38
Ratios of precursor subsets in human marrow, proliferative indices, and
differentiation were evaluated in the present study with the goal of
identifying age-related changes. We now report that whereas B-cell
precursors are depressed in some patients, the entire differentiation
series is normally present throughout life and is mitotically active.
However, surface marker changes occur early in life, and adult marrow
has reduced potential for lymphocyte production in 2 model systems.
Additional information about acquisition of CD7 and CD10 may prove
helpful in understanding the sequence of early differentiation events
in humans.
Cell sources
Animals
Antibodies and cytokines The following antibodies were either purified, biotinylated, or conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC): anti-CD38 (clone HIT2), anti-CD24 (clone ML5), anti-CD23 (clone M-L233), anti-CD45 (clone HI30), anti-Ki-67 (clone B56), anti-IgD (clone IA6-2) from BD-Pharmingen; (San Jose, CA), anti-CD34 (HPCA-2), anti-CD3 (clone UCHT1), anti-CD7 (clone 6B7), anti-CD8 (clone HIT8a), anti-CD14 (clone TUK4), anti-CD16 (clone 3G8), anti-CD10 (clone 5-1B4), anti-CD19 (clone SJ25-C1), anti-CD13 (clone TUK1), anti-CD27 (clone CLB-27/1), anti-CD33 (clone 4D3), anti-CD56 (clone NKI nbl-1), anti-CD64 (clone 10.1), anti- light
chain (clone HP6062), anti- light chain (clone HP6054), and
anti-glycophorin A (clone CLB-ery1) from Caltag Laboratories (Burlingame, CA). Fab' fragments of goat antihuman IgM or
antihuman IgD were from Southern Biotechnology Associates (Birmingham,
AL). The biotinylated antibodies were revealed with streptavidin red 613 (Gibco BRL, Rockville, MD). Purified recombinant human (rh) erythropoietin (rhEPO), rh stem cell factor (rhSCF), rhFlt3 ligand, rhIL-15, and rh granulocyte colony-stimulating factor (rhG-CSF) were
purchased from R & D Systems (Minneapolis, MN).
Cell isolation and staining procedure for fluorescence activated cell sorting analysis Four-color immunofluorescence analysis was used for the identification of the different B-cell precursor populations in total nucleated BM cell suspensions from fetal and adult marrow. Briefly, single-cell suspensions were obtained by flushing or gently vortexing the BM. After staining, erythrocytes were lysed using the Becton Dickinson lysis buffer solution according to the manufacturer's instructions and CD45 antibody was used to assess the percentages of leukocytes in each sample. Intracellular staining was done by permeabilizing cells after surface staining. The cells were fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 70% ethanol at 20°C, for more than 30 minutes
and washed twice with the staining buffer. They were then incubated
with antihuman IgM for 30 minutes at room temperature. To establish the
proliferative fraction, cells were incubated with the antibody
anti-Ki67 (mib-1) for 1 hour on ice, as described by Zupo et
al.39 Data acquisition and analyses were performed on a
FACSCalibur (Becton Dickinson).
Isolation of CD34+/  CD10CD7,
CD34+LinCD10CD7+,
and
CD34+LinCD10+CD7
cells were sorted using a MoFlo (Cytomation, Fort Collins, CO). The
sorted cells were subsequently subjected to flow cytometry, culture,
and gene expression analyses. In some experiments,
CD34+CD38 cells were sorted and cultured. In
this case, mononuclear cells were incubated with mouse antihuman
antibodies: CD3, CD13, CD14, CD16, CD33, CD56, and glycophorin A, and
negatively selected using the BioMag goat antimouse IgG-coated beads
(Perseptive Biosystems, Framingham, MA). The enriched cells were
stained with CD34-FITC and CD38-PE. CD34+CD38
cells were determined using gating previously described.40 Analysis after sorting revealed the purity of the sort populations to
be more than 95%.
Transplantation of human cells into NOD/SCID mice Magnetically enriched CD34+ cells from CB or fetal or adult BM were diluted in PBS with 0.1% bovine serum albumin (BSA fraction V; Sigma Chemical, St Louis, MO) and injected into the tail vein of sublethally irradiated (100 cGy from a 137Cs source) 8- to 12-week-old male or female NOD/SCID mice. Then, 5 × 106 CD34 CB mononuclear cells were
coinjected as a source of accessory cells.41,42 No
chimerism was ever obtained in mice given transplants of the accessory
cells alone.
LDAs The frequencies of B-cell progenitors in CB and adult BM were determined by plating CD34+CD38 cells in
limiting dilution assays (LDAs). Between 20 and 180 wells containing
pre-established MS-5 stromal cell layers (a kind gift of Dr J. Mori,
Niigata University) were plated with 3, 5, 10, 15, 20, 30, 50, 75, 100, 150, or 200 cells each using the automated cell deposition unit of the
MoFlo. Cells were cultured in  -minimal essential medium
(MEM; Cellgro-Mediatech) supplemented with 10% fetal calf serum (FCS;
Hyclone, Logan, UT) and a combination of rhSCF (100 ng/mL) and rhG-CSF
(10 ng/mL).21 Wells were inspected every week for the
presence of new clones. Positive wells were harvested after 5 to 7 weeks of culture and analyzed by flow cytometry for CD19+
cells. The frequencies of precursors were calculated by linear regression analysis on the basis of Poisson distribution as the reciprocal of the concentration of test cells that gave 37%
negative cultures.
Multilineage coculture system and methycellulose culture Sorted CD34+LinCD10CD7,
CD34+LinCD10CD7+,
and
CD34+ LinCD10+CD7
cells were cultured at 1 × 104 cells/6
mL/25-cm2 tissue culture flask (Corning, Corning, NY) with
MS-5 stromal cells. Culture media was -MEM containing 10% FCS,
rhSCF (100 ng/mL), rhG-CSF (10 ng/mL), rhFlt-3 ligand (100 ng/mL), and
rhIL-15 (10 ng/mL). The cultures were maintained with weekly whole
medium changes and the generation of myeloid, B, and NK cells was
evaluated at the indicated periods. Methylcellulose cultures were
performed in 35-mm plates (Corning) using MethoCult GF H4534 medium
(Stem Cell Technologies, Vancouver, BC, Canada) containing rhSCF, rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF), and rhIL-3
along with 3 U/mL rhEPO (R & D Systems). All cultures were maintained
at 37°C in a humidified incubator with 5% CO2 in air. Differential colony counts were scored after 10 to 14 days by morphologic characteristics using an inverted microscope and confirmed by staining individual colonies with Wright or
May-Grünwald-Giemsa stain.
RT-PCR Sorted cells were put in lysis buffer (Ambion, Austin, TX) and mRNA was extracted using Poly-A column (Ambion) according to the manufacturer's instructions. cDNA was prepared from mRNA treated with DNase I using oligo-dT and Moloney murine leukemia virus reverse transcriptase (RT; Gibco BRL). Semiquantitative polymerase chain reaction (PCR) was done to measure relative differences in transcript levels of target cDNAs against levels of the reference gene GAPDH. The PCR was done in 100 µL containing PCR buffer (Takara Biomedical, Osaka, Japan), 1.5 mM MgCl2, 200 µM deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), 50 µM deoxycytidine triphosphate (dCTP), and 50 pmol of each primer. For quantification, 0.5 µCi (0.0185 MBq) [32P] dCTP (Amersham, Arlington Heights, IL) was
included in each reaction tube. Samples were denatured in a DNA
thermocycler (Perkin-Elmer, Norwalk, CT) for 10 minutes at 95°C. To
increase specificity, 2.5 U Taq DNA polymerase (Takara) was
added to each sample during this initial denaturation. Samples were
then cycled for 1 minute at 94°C, 1 minute annealing 60°C, 1.5 minutes extension at 72°C with a final extension of 7 minutes at
72°C. Aliquots were removed at cycles 25, 28, 31, and 34 for
glyceraldehyde phosphate dehydrogenase (GAPDH) and cycles 32, 35, and
38 for all others to ensure that PCR remained within the exponential
range of amplification. Then, 5-µL aliquots were denatured in
a formamide loading buffer and applied to a 6% polyacrylamide gel
containing 7 M urea. Incorporation of [alpha32P] dCTP
into PCR product bands was quantified from dry gels using a
PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
Primers were as follows: GAPDH sense: 5'-TCCAAAATCAAGTGGGGCGAT-3', GAPDH antisense: 5'-TTCTAGACGGCAGGTCAGGTC-3', 475-bp expected product; RAG1 sense: 5'-CCTGAGTCCTCTCATTGCTGAGAG-3', RAG1 antisense: 5'-AGGGCATGATGATCGCCATACT-3', 681-bp product; RAG2 sense: 5'-CTAATGAAGAGCAGACAACATTCA-3', RAG2 antisense: 5'-TAGGACTCTTTGGGGAGTGTGTAG-3', 422-bp product; EBF sense: 5'-CCGGGCTCACTTTGAGAAGCAG-3', EBF antisense: 5'-CAGGGAGTAGCATGTTCCAGAT-3', 638-bp product; Pax5 sense: 5'-CTCGGTGAGCACGGATTCGGCC-3', Pax5 antisense: 5'-GCGGCAGCGCTATAATAGTAG-3', 621-bp product; CD122 sense: 5'-GCGTGGCTCGGCCACCTC-3', CD122 antisense: 5'-GACGATGAGGGGAAGGGCGAAGA-3', 211-bp product, Id-1 as described.43 Statistical analysis All results are shown as mean values ± SD. Differences between groups were assessed using the Student t test.
B lymphopoiesis persists throughout adult human life It has been reported that absolute numbers of B-lymphocyte precursors in human marrow decline with normal age and particularly during adolescence.38 Therefore, we calculated percentages of CD19+sIg (surface Ig) lymphocytes and
CD19+sIg+ B cells relative to total numbers of
nucleated cells in our adult marrow samples (data not shown). As in the
previous study,38 sample-to-sample variations were
considerable, but we saw no substantial or consistent depletion of
these lymphocyte populations as a function of age in adults. We then
extended previous studies31,35 describing B-lineage
lymphocyte precursors in adult human marrow (Figure 1A). Pro-B cells
(CD34+CD10+CD19+), pre-B cells
(CD34CD19+sIg or
CD19+cµ+sIg) and
immature B cells
(sIgM+CD24hiIgDCD10+CD38+)
represented relatively constant percentages of the CD19+
lymphocytes over a 24- to 88-year age range. The ratios
between these populations and the presence of naive
(sIgMhiIgD+CD24hiCD10+/CD38+/CD27)
B cells would all be consistent with stable B lymphopoiesis. Mature
recirculating B cells
(sIgM+IgD+CD24loCD38)
were previously shown to comprise a large fraction of marrow lymphocytes.35 We found no mature B cells in fetal bones,
but there was no substantial influence of adult age on this population (Figure 1A). Mature B cells in marrow were also CD27+, a
characteristic of somatically mutated memory B cells in peripheral blood.44 Most female marrow donors were receiving estrogen
replacement therapy, and we found no sex-related differences in
this analysis.
Blood cell production results from massive division within marrow, and we assessed expression of the Ki-67 nuclear proliferation antigen in B-lineage lymphocyte precursors (Figure 1B). Again, we found no age-related changes in this parameter and male-versus-female values were comparable. A similar analysis was recently performed with fetal marrow specimens,45 and the results are included here for comparison (Figure 1B). With the exception of small pre-B cells, which are more mitotically active during embryonic life, B-cell precursors were remarkably similar with respect to Ki-67 expression. We conclude that actively proliferating precursors are present within the lymphocyte fraction of human marrow until at least the eighth decade. Analysis of marrow from exceptional patients reveals useful indices of B-cell production The present data would be consistent with stable B lymphopoiesis occurring over a large age range. However, it is difficult to know the degree to which lymphopoietic marrow is replaced by fat as a function of age and to control for sample-to-sample variations with respect to peripheral blood contamination and location. Therefore, it would be helpful to have other parameters for assessing marrow activity. Relatively constant proportions of B-lineage lymphocytes were found in most of the specimens we examined (Figure 1A). Whenever immature (IgM+CD24hiIgDCD10+CD38+)
B cells were present, marrow samples contained mitotically active pre-B
(CD19+sIgM) cells identified with the Ki-67
antibody. However, exceptionally low numbers were found in samples
obtained from some patients. There were insufficient numbers of these
individuals and information about specific treatments to formally
assign them to groups. However, the degree of sample-to-sample
variation in these specimens provided an opportunity to seek
correlations (Figure 2). In these
individuals, incidences of cycling pre-B cells predicted the presence
of immature lymphocytes (P = .0001), consistent with a
close precursor-product relationship between them. Naive
(IgM+CD24hiIgD+CD38+CD10+/D27)
B cells did not closely correlate with proliferating pre-B cells (P = .1035), although they are the progeny of immature B
cells. We emphasize the usefulness of Ki-67 expression because simple percentages of pre-B cells did not correspond as closely with numbers
of newly formed lymphocytes (data not shown). The findings suggest that
the size of the proliferating pre-B cell pool determines numbers of
newly formed B cells.
Early developmental age-related changes in populations resolved by flow cytometry Earlier reports demonstrated that CD7 could be used to discriminate hematopoietic stem cells from more differentiated precursors of NK and T lymphocytes.46,47 It is also noteworthy that CD7 is rarely coexpressed with CD10 or CD19, and evidence indicates that CD7+ cells in CB represent common lymphoid progenitors.33 However, comparisons made by flow cytometry suggested that CB might not be representative of differentiation pathways used in adult marrow. Adult CD34+Lin cells contained a more conspicuous
subset of CD7CD10+ progenitors as well as
more CD7+CD10 cells (Figure
3A). Additional differences were found by
comparing sorted CD7+ and CD7 subsets of
CD34+LinCD10 cells from the 2 sources (Figure 3B-C). It has been suggested that CD45RA is acquired
before CD10,32 and we found that nearly half of the CB
CD7+ cells expressed moderate to high levels (Figure 3B).
In contrast, only 10% of the comparable subset from adult marrow was
CD45RA+. As noted above, the CD38 subset of
the CD34+LinCD7+ fraction of CB
has been recently found to contain multilymphoid but not myeloerythroid
progenitors.33 It is remarkable that cells with that
combination of markers do not exist in adult marrow (Figure 3C). Note
that the CD7 subsets of fetal and adult populations were
similar with respect to CD45RA and CD38 expression. Thus, the
composition of hematopoietic cell subsets or surface marker display in
CB differs substantially from adult BM.
Transplantation experiments reveal age-related changes in differentiation potential Immunodeficient NOD/SCID mice were prepared for transplantation with low-dose irradiation ("Materials and methods") and then injected with highly enriched suspensions of CD34+ cells (Table 1). Previous studies documented an extraordinary differentiation potential of CB stem cells in this model,48-50 so we compared one log less CD34+ cells from this source to ones isolated from fetal and adult marrow. The latter represented pools of CD34+ cells obtained from specimens ranging from 35 to 82 years of age in one experiment and 62 to 77 years of age in a second transplantation. Flow cytometry was performed on aliquots of the suspensions to determine numbers of CD34+CD38 cells injected because
of reports that these cells are particularly effective for engraftment
of NOD/SCID mice51 (Table 1). It has been shown that
undefined accessory cells can influence engraftment of human cells in
mice.41,42 Therefore, we added 5 × 106
CD34 CB mononuclear cells to all samples of
CD34+ cells before transplantation, and the recipients were
assessed 7 weeks later. No human cells were detectable in animals that received only the CD34 cells, but easily discernible
populations of CD45+CD19+ lymphocytes were
found in virtually all recipients of CD34+ cells,
regardless of donor age (Table 1). Adult precursors were less effective
than those obtained from the other 2 sources with respect to numbers of
lymphocytes produced, suggesting a possible consequence of the early
developmental age-related changes noted.
Culture experiments reveal age-related differences in expansion and differentiation potential of lymphocyte progenitors Homing to the marrow, resistance to xenotransplantation barriers, and many other factors could influence the degree of chimerism observed in the NOD/SCID model. Furthermore, culture experiments could permit detailed analysis of small cell numbers. Therefore, we performed an LDA in which graded numbers of CD34+CD38 cells
were seeded onto monolayers of murine MS-5 stromal cells and stimulated
with SCF and G-CSF (Table 2). The
frequency of precursors with the potential for clonal expansion varied
from 3.6% to 9.78% in marrow suspensions obtained from healthy 24- to
88-year-old donors. In separate experiments, the cloning efficiency of
umbilical CB CD34+CD38 cells ranged from
11.11% to 14.29% using the same culture conditions. In this case, the
B-cell progenitor frequency was 6.67% (range, 5.56%-8.33%).
Furthermore, in about 40% of the wells more than 50% of the cells
produced by CB CD34+CD38 cells were
CD19+, and this was never the case with adult BM. Analysis
of pooled clones revealed that differentiation did not proceed
efficiently beyond the CD19+ stage in such cultures and
very few cytoplasmic or surface µ+ cells were
recovered.
Culture conditions were then developed that allowed simultaneous
differentiation of CD19+ B- lineage, CD13/CD33+
myeloid-lineage, and CD56+ NK-lineage cells ("Materials
and methods"). Absolute numbers of CD19+ cells produced
were equal to or more than those obtained with other methods we have
tried and NK-lineage cells were always efficiently produced when IL-15
was present. The method was used for a side-by-side comparison of
fetal/neonatal-versus-adult marrow precursors. Three-week cultures
initiated with 104
CD34+Lin
Myeloid potential was highest in the
CD34+Lin Weekly examination revealed that myeloid progeny appeared quickly and
then declined in cultures initiated with the
CD34+ Lin Neonatal or adult CD10+ fractions contained B-lineage precursors that differentiated within 2 weeks in culture, and CD56+ NK-lineage cells reached peak values 1 week later. This analysis revealed large age-related changes in the potential of highly purified CB and adult BM progenitors to expand in culture. CD10 and CD7 expression denote commitment to B and NK lineages These findings highlight the importance of learning more about the B-lineage precursors in human marrow. Therefore, adult subsets sorted for use in the culture experiments as described were also subjected to RT-PCR analysis (Figure 5). Transcripts associated with B-lymphocyte lineage differentiation (RAG1, RAG2, EBF, and Pax5) were markedly up-regulated in the CD34+LinCD10+CD7
population relative to the more primitive
CD34+LinCD10CD7
subset. Furthermore, levels of the Id-1 transcriptional repressor were
reduced in that fraction (data not shown). In contrast, expression of
the CD122 receptor for IL-15/IL-2 required by NK-lineage cells was
strongly associated with display of CD7. Although transcripts for
RAG1 and RAG2 were detectable in CD7+ cells, this
fraction was deficient in the EBF transcription factor required for B
lymphopoiesis. Very similar results were obtained in 3 independent
experiments. These results complement those obtained with culture
experiments and suggest that acquisition of CD10 or CD7 corresponds to
substantial progression in the B or NK lineages, respectively.
This study was informative about several aspects of human B lymphopoiesis. Ratios between precursors and immature B cells within marrow and an indication of mitotic activity were found to be remarkably constant throughout adult life. Exceptional marrow samples from some patients were deficient in lymphocyte precursors, providing a unique opportunity to see how the abundance of particular subsets corresponds to newly formed B cells. Although the potential for lymphoid differentiation, as assessed by culture and transplantation assays was retained in most individuals, the lymphocyte yield from adult progenitors was much less than that obtained from CB. Furthermore, we documented age-related changes in subsets resolved on the basis of CD7 and CD10 expression. Analysis of highly purified progenitors revealed that erythroid and NK differentiation potential might diverge from B-lineage potential as cells acquire one or the other of these markers. There are many technical problems associated with analysis of human marrow specimens, and it is difficult to estimate absolute numbers of B-cell precursors. As one example, mature recirculating B cells have been identified that have a distinctive density of CD24 and sIgM.35 Absolute numbers of these cells per unit marrow or body weight are not known, but they progressively dilute B-cell precursors and newly formed B cells. However, it is fortunate that other measurements reflect the lymphopoietic activity in that organ. As in previous studies31,35 the ratios between B-cell precursors and newly formed B cells can be determined, and we confirm that they are relatively constant. Moreover, the proliferative activity of B-cell precursors was surprisingly stable with age. In a separate study, we evaluated precursors in fetal marrow and found that an exceptionally large fraction of small pre-B cells in that site are Ki-67+.45 This is one of several distinctive features of fetal lymphopoiesis. Once the process is established in adult marrow, the proliferation index of B-cell precursors remains unchanged until at least 88 years of age. The striking deficiency of B-cell precursors in some of the marrow
specimens suggests that influences of therapy and environmental stress
on B lymphopoiesis merit further study. One previous report recorded
delayed recovery of B cells after marrow transplantation in patients
receiving cortisone.52 Most of our deficient samples came
from patients who were being treated with anti-inflammatory drugs and
we will show elsewhere that human lymphocyte progenitors are very
sensitive to glucocorticoids. Here we used the variation provided by
these unusual specimens to make inferences about precursor-product relationships within marrow. The best correlation was found between cycling pre-B cells and immature B cells. The Ki-67 proliferation antigen provided a particularly useful index, and simple pre-B cell
percentages did not correlate as well. Whenever percentages of
proliferating pre-B cells were beneath 50%, immature
(IgM+IgD It was important to learn if cells with surface characteristics of
B-cell precursors actually had the potential to give rise to
lymphocytes. Human CD34+CD38 Limiting dilution cultures initiated with highly purified
CD34+CD38 Because the proportion of hematopoietic marrow relative to fat
progressively declines with age in long bones, total numbers of newly
formed B cells might be reduced in some locations.28 However, the cellularity of marrow in other sites is remarkably constant throughout life, and it will be important to determine the
total body output of new B cells.29 This important issue awaits other types of analyses comparable to those recently used to
assess thymic activity in adult humans.55-57 Many studies
suggest that hematopoietic stem cells retain the potential for
replenishing lymphocytes throughout life.58,59 However,
there are changes with respect to incidence, homing potential, and
function on a per-cell basis. Additionally, there can be age-related
declines in the ability of environmental cells within marrow to support lymphopoiesis.4 Genetic polymorphisms influence stem cell
numbers and senescence to a substantial degree.59,60 For
that reason, our culture assays were conducted with cells from
individual marrow donors. Although flow cytometry revealed proportions
of lymphocyte precursors in most marrow specimens to be remarkably
constant, functional precursor frequencies among
CD34+CD38 Flow cytometry experiments revealed that the composition and patterns
of surface marker expression differ substantially between CB and adult
BM. The interesting
CD34+CD38 The question arises whether B lineage-associated events begin prior to
the CD10+ stage and CD7+ cells are interesting
in this regard. One recent study found that a
CD34+CD38 To summarize, several distinctive adult characteristics of
B-lymphocyte precursors may be acquired during the neonatal or adolescent period. The primitive
CD34+Lin
Submitted March 21, 2002; accepted August 14, 2002.
Prepublished online as Blood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-03-0896.
Supported by grants AI 20069 and AI 45864 from the National Institutes of Health. P.W.K. holds the William H. and Rita Bell Chair in biomedical research.
M.I.D.R. and T.Y. 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: Paul W. Kincade, Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104; e-mail: kincade{at}omrf.ouhsc.edu.
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© 2003 by The American Society of Hematology.
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  D. Frasca, A. M. Landin, R. L. Riley, and B. B. Blomberg Mechanisms for Decreased Function of B Cells in Aged Mice and Humans J. Immunol., March 1, 2008; 180(5): 2741 - 2746. [Abstract] [Full Text] [PDF]
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M. E. Hystad, J. H. Myklebust, T. H. Bo, E. A. Sivertsen, E. Rian, L. Forfang, E. Munthe, A. Rosenwald, M. Chiorazzi, I. Jonassen, et al. Characterization of Early Stages of Human B Cell Development by Gene Expression Profiling J. Immunol., September 15, 2007; 179(6): 3662 - 3671. [Abstract] [Full Text] [PDF]
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S. E. Johnson, N. Shah, A. Panoskaltsis-Mortari, and T. W. LeBien Murine and Human IL-7 Activate STAT5 and Induce Proliferation of Normal Human Pro-B Cells J. Immunol., December 1, 2005; 175(11): 7325 - 7331. [Abstract] [Full Text] [PDF]
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G. R.A. Ehrhardt, J. T. Hsu, L. Gartland, C.-M. Leu, S. Zhang, R. S. Davis, and M. D. Cooper Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells J. Exp. Med., September 19, 2005; 202(6): 783 - 791. [Abstract] [Full Text] [PDF]
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H. Igarashi, K. L. Medina, T. Yokota, M. I. D. Rossi, N. Sakaguchi, P. C. Comp, and P. W. Kincade Early lymphoid progenitors in mouse and man are highly sensitive to glucocorticoids Int. Immunol., May 1, 2005; 17(5): 501 - 511. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), CD34+Lin
), and
CD34+Lin
) fractions sorted from adult BM. RT-PCR was used to amplify
transcripts for the indicated genes, and the results were normalized
according to GAPDH expression.
.
J Immunol.
1989;142:3875-3883