developmental age related changes (Running title: Age related changes in human B lymphopoiesis)

doi:10.1182/blood-2002-03-0896Prepublished online August 29, 2002;

and Paul W KincadeMaria Isabel D Rossi, Takafumi Yokota, Kay L Medina, Karla P Garrett, Philip C Comp, Arthur H Schipul developmental age related changesB lymphopoiesis is active throughout human life, but there are

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1

B lymphopoiesis is active throughout human life, but there are

developmental age related changes

(Running title: Age related changes in human B lymphopoiesis)

Maria Isabel D. Rossi*#, Takafumi Yokota*#, Kay L. Medina*, Karla P. Garrett*, Philip C.

Comp¶, Arthur H. Schipul, Jr.† and Paul W. Kincade*

*Immunobiology & Cancer Program, Oklahoma Medical Research Foundation

825 N.E. 13th Street, Oklahoma City, OK 73104

¶Department of Medicine, University of Oklahoma Health Sciences Center

Oklahoma City, OK 73104

†St. Anthony Hospital, Oklahoma City, OK 73101

#The first two authors contributed equally to this work.

Scientific heading: Immunobiology

This work was supported by grants AI 20069 and AI 45864 from the National Institutes

of Health.

Address correspondence to:

Paul W. Kincade, Immunobiology & Cancer Program, Oklahoma Medical Research

Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104

Tel: (405) 271-7905, fax: (405) 271- 8568, [email protected]

Word counts: Abstract 237 words / total text 5404 words

Copyright (c) 2002 American Society of Hematology

Blood First Edition Paper, prepublished online August 29, 2002; DOI 10.1182/blood-2002-03-0896 For personal use only. by guest on June 2, 2013. bloodjournal.hematologylibrary.orgFrom



2

Abstract

This study addressed several questions concerning age related changes in

human B lymphopoiesis. The relative abundance of pro-B, pre-B, immature, naïve 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- CD10+

progenitors from cord blood and adult marrow had equivalent potential for differentiation

in culture and notable age related changes were found in more primitive subsets. A

recently described subset of CD34+ CD38- CD7+ cord blood cells had no exact

counterpart in adult marrow. That is, all adult CD34+ Lin- CD7+ CD10- cells expressed

CD38, displayed less CD45RA and had little B lineage differentiation potential. The

CD7+ fractions in either site contained progenitors for erythroid and NK lineages and

ones sorted from marrow expressed high levels of transcripts for the CD122 IL-2/IL-15

receptor required by NK lineage precursors. Dramatic changes in human B

lymphopoiesis occur early in life and more information is required to construct a

probable sequence of differentiation events prior to the acquisition of CD10.

Corresponding Author: [email protected]




3

Introduction

Humoral immune responses are known to be compromised in the aged, and

decreased numbers of B and T lymphocytes parallel increases in NK cells.1-3 Animal

studies have revealed age related changes in B lymphocyte production within bone

marrow.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 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-c-

kitLoTdT+IL-7Rα+/Lo precursors likely derive from early lymphoid progenitors in the very

rare Lin-c-kitHiSca-1HiCD27+ fraction of bone marrow cells.14,15 However, it is important




4

to note that the differentiation pathways utilized during fetal life may be quite different

from adult bone marrow.16-18

Knowledge of early B lymphocyte progenitors in humans lags behind that for

mice, and there are important differences. 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, there is evidence that

CD10+CD19- cells may be the human equivalent of common lymphoid progenitors in the

mouse, while candidates recently identified in umbilical cord blood are CD34+CD38-

CD7+.32,33 A major goal of this study was to determine if numbers and functions of such

precursors decline with age, because that could reflect important changes in human

lymphopoietic capacity.

Expression of Rag genes in marrow of mature individuals might suggest that Ig

genes are being rearranged.34,35 Unfortunately, little information has been available




5

about proliferation36,37 or differentiation activity in humans, and it is possible this slows

with age. Thus, while 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 while 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 two model systems.

Additional information about acquisition of CD7 and CD10 may prove helpful in

understanding the sequence of early differentiation events in humans.




6

Materials and Methods

Cell Sources. Cord blood samples were obtained from placentas of healthy newborns

collected at the Oklahoma University or Saint Anthony Hospitals (Oklahoma City, OK).

Adult marrow samples were collected from patients between 24- to 88-years of age that

were undergoing hip replacement surgery at the Bone and Joint Hospital (Oklahoma

City, OK). Patient records with no identifying information were used to determine that

some individuals had a recent history of treatment with anti-inflammatory drugs such as

prednisone or the drug Naprosyn. Most specimens came from otherwise healthy

patients with no evidence of malignancy or immunosuppression. The Anatomic Gift

Foundation (Laurel, MD) supplied femoral bone marrow from fetuses between 16 and

24 weeks of gestation. An Investigational Review Board approved all procedures

involving human specimens.

Animals. NOD/LtSz-scid/scid (NOD/SCID) mice were obtained from a breeding colony

established at the Oklahoma Medical Research Foundation (OMRF, Oklahoma City,

OK) from breeding pairs kindly provided by Dr. Leonard D. Schultz (The Jackson

Laboratory, Bar Harbor, ME). Animals were housed in a restricted barrier facility under

specific pathogen-free conditions.

Antibodies and Cytokines. The following antibodies were either purified, biotinylated or

conjugated with 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 PharMingen, (Becton & Dickinson, San




7

Jose, CA), anti-CD34 (HPCA-2) from BD (Becton & Dickinson), 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-κ light chain (clone HP6062), anti-λ light chain

(clone HP6054), and anti-glycophorin A (clone CLB-ery1) from Caltag Lab. (Burlingame,

CA). F’(ab) fragments of goat anti-human IgM or anti-human IgD were from Southern

Biotechnology Associates Inc. (Birmingham, AL). The biotinylated antibodies were

revealed with Streptavidin Red 613 (Gibco, BRL, Rockville, MA). Purified

rherythropoietin (EPO), rhSCF, rhFlt3 ligand, rhIL-15 and rhG-CSF were purchased

from R & D Systems (Minneapolis, MN).

Cell isolation and staining procedure for FACS analysis. Four- color

immunofluorescence analysis was used for the identification of the different B cell

precursor populations in total nucleated bone marrow cell suspensions from fetal and

adult marrow. Briefly, single cell suspensions were obtained by flushing or gently

vortexing the bone marrow. After staining, erythrocytes were lysed using the B-D 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 PBS and permeabilized with 70% ethanol at –20ºC, for more than

30 minutes and washed twice with the staining buffer. They were then incubated with

anti-human IgM for 30 min at room temperature. To establish the proliferative fraction,




8

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+ cells and cell sorting. Mononuclear cells were collected from cord

blood or adult marrow by Ficoll/Hypaque (Lymphocyte Separation Medium, Cellgro-

Mediatech, Herndon, VA) centrifugation. Enrichment for CD34+ cells was performed

using the Direct CD34 Isolation Kit (Miltenyi Biotechnology (Auburn, CA). CD34+

enriched cells were stained with CD34-FITC, lineage markers (Lin, CD13, CD14, CD33

and CD64 for myeloid cells, glycophorin A for erythroid cells, CD19 for B cells, CD3 and

CD8 for T cells, CD56 for NK cells)-PE, CD7-biotin followed by Streptavidin Red613,

and CD10-APC. CD34+Lin-CD10-CD7-, CD34+Lin-CD10-CD7+, and CD34+Lin-

CD10+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 anti-human antibodies: CD3,

CD13, CD14, CD16, CD33, CD56 and glycophorin A, and negatively selected using the

BioMag goat anti-mouse IgG-coated beads (PerSeptive Biosystems Inc., Framingham,

MA). The enriched cells were stained with CD34-FITC and CD38-PE. CD34+CD38-

cells were determined using gating previously described.40 Post-sort analysis revealed

the purity of the sort populations to be greater than 95%.




9

Transplantation of human cells into NOD/SCID mice. Magnetically enriched CD34+

cells from cord blood, fetal or adult marrow were diluted in PBS with 0.1% BSA (bovine

serum albumin fraction V, Sigma Chemical Co.) and injected into the tail vein of sub-

lethally irradiated (100 cGy from a 137Cs source) 8- to 12-weeks old male or female

NOD/SCID mice. 5 x 106 CD34- cord blood mononuclear cells were co-injected as a

source of accessory cells.41,42 No chimerism was ever obtained in mice transplanted

with the accessory cells alone.

Limiting dilution assays (LDA). The frequencies of B cell progenitors in cord blood and

adult marrow were determined by plating CD34+CD38- cells in limiting dilution assays.

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 α-MEM (Cellgro-Mediatech) supplemented with 10% 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.

Multil-lineage co-culture system and methycellulose culture. Sorted CD34+Lin-CD10-

CD7-, CD34+Lin-CD10-CD7+, and CD34+Lin-CD10+CD7- cells were cultured at 1 x 104




10

cells per 6 mL/25-cm2 tissue culture flask (Corning, NY) with MS-5 stromal cells.

Culture media was α-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 35mm

plates (Corning) using MethoCultTM GF H4534 medium (StemCell Technologies Inc.,

Vancouver, Canada) containing rhSCF, rhGM- CSF and rhIL3 along with 3 units/ml of

rhEPO (R&D Systems). All cultures were maintained at 37ºC in a humidified incubator

with 5% CO2 in air. Differential colony counts were scored after 10-14 days by

morphological characteristics using an inverted microscope and confirmed by staining

individual colonies with Wright’s or May-Grunwald-Giemsa stain.

Reverse transcriptase polymerase chain reaction (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 Dnase

I-treated mRNA using oligo-dT and Moloney murine leukemia virus reverse

transcriptase (Gibco BRL). Semiquantitaive PCR was done in order to measure relative

differences in transcript levels of target cDNAs against levels of the reference gene

GAPDH. The PCR reation was done in 100 µl containing 1 X PCR buffer (TaKaRa

Biomedical, Osaka, Japan), 1.5mM MgCl2, 200 µM dATP, dGTP, dTTP, 100 µM dCTP,

and 50 pmoles of each primer. For quantification, 0.5 µCi [alpha32P] dCTP (Amersham,

Arlington Heights, IL) was included in each reaction tube. Samples were denatured in a

DNA thermocycler (Perkin-Elmer, Norwalk, CT) for 10 min at 95ºC. To increase




11

specificity, 2.5 U of Taq DNA polymerase (TaKaRa) was added to each sample during

this initial denaturation. Samples were then cycled for 1 min at 94ºC, 1 min annealing

60°C,1.5 min extension at 72ºC with a final extension of 7 min at 72ºC. Aliquots were

removed at cycles 25, 28, 31 and 34 for GAPDH and cycles 32, 35 and 38 for all others

to insure that PCR remained within the exponential range of amplification. 5 µl aliquots

were denaturated 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,

RAG-sense: 5’-CCTGAGTCCTCTCATTGCTGAGAG-3’, RAG1-antisense: 5’-

AGGGCATGATGATCGCCATACT-3’, 681bp product , RAG-2-sense: 5’-

CTAATGAAGAGCAGACAACATTCA-3’, RAG2-antisense: 5’-

TAGGACTCTTTGGGGAGTGTGTAG-3’, 422bp product, EBF-sense: 5’-

CCGGGCTCACTTTGAGAAGCAG-3’, EBF-antisense: 5’-

CAGGGAGTAGCATGTTCCAGAT-3’, 638bp product, PAX-5-sense: 5’-

CTCGGTGAGCACGGATTCGGCC-3’, PAX-5-antisense: 5’-

GCGGCAGCGCTATAATAGTAG-3’, 621bp product, CD122-sense: 5’-

GCGTGGCTCGGCCACCTC-3’, CD122-antisense: 5’-

GACGATGAGGGGAAGGGCGAAGA-3’ ,211bp product, Id-1 as described.43

Statistical analysis. All results are shown as mean values ± S.D. Differences between

groups were assessed using the Student t test.




12

Results

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- 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 (CD34-

CD19+sIg- or CD19+cµ+ sIg-) and immature B cells (sIgM+CD24hiIgD-CD10+CD38+)

represented relatively constant percentages of the CD19+ lymphocytes over a 24-88

year age span. The ratios between these populations and the presence of naïve (sIgMhi

IgD+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.




13

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, that 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 data shown above 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 above). Whenever immature (IgM+CD24hiIgD-CD10+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




14

opportunity to seek correlations (Figure 2). In these individuals, incidences of cycling

pre-B cells predicted the presence of immature lymphocytes (p = 0.0001), consistent

with a close precursor-product relationship between the two. Naïve (IgM+CD24hiIgD+

CD38+CD10+/- D27-) B cells did not closely correlate with proliferating pre-B cells (p =

0.1035), 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 co-expressed with CD10 or CD19, and there is evidence

that CD7+ cells in cord blood represent common lymphoid progenitors.33 However,

comparisons made by flow cytometry suggested that cord blood might not be

representative of differentiation pathways utilized in adult marrow. Adult CD34+ Lin-

cells contained a more conspicuous subset of CD7-CD10+ progenitors as well as more

CD7+CD10- cells (Figure 3A). Additional differences were found by comparing sorted

CD7+ and CD7- subsets of CD34+Lin-CD10- cells from the two sources (Figure 3B and

3C). It has been suggested that CD45RA is acquired before CD1032 and we found that

nearly half of the cord blood CD7+ cells expressed moderate to high levels (Figure 3B).

In contrast, only 10% of the comparable subset from adult marrow was CD45RA+. As




15

noted above, the CD38- subset of the CD34+ Lin- CD7+ fraction of cord blood has been

recently found to contain multi-lymphoid, but not myelo-erythroid 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 and/or surface marker display in cord blood differs substantially from adult

marrow.

Transplantation experiments reveal age related changes in differentiation potential.

Immunodeficient NOD/SCID mice were prepared for transplantation with low dose

irradiation (see Materials & Methods) and then injected with highly enriched

suspensions of CD34+ cells (Table 1). Previous studies documented an extraordinary

differentiation potential of cord blood 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 transplant. 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 x 106 CD34- cord blood 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




16

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 two sources with respect to numbers of lymphocytes produced,

suggesting a possible consequence of the early developmental age related changes

noted above.

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 a limiting

dilution assay where 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 normal healthy 24-88 year old

donors. In separate experiments, the cloning efficiency of umbilical cord blood 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% (5.56% to 8.33%). Furthermore, in

about 40% of the wells more than 50% of the cells produced by cord blood CD34+CD38-

cells were CD19+, and this was never the case with adult marrow. 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




17

of CD19+ B lineage, CD13/CD33+ myeloid lineage, and CD56+ NK lineage cells (see

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-CD7-CD10- cord blood precursors yielded much

larger numbers of B lineage lymphocytes than those prepared with equivalent cells from

adult marrow (Figure 4A). Interestingly, the lymphoid potential of CD34+Lin-CD7-

CD10+ cells from the sources was comparable. The CD34+Lin-CD7+CD10- subset was

efficient only when derived from cord blood. All subsets of cord blood yielded

approximately 10 times the number of CD56+ NK lineage cells than obtained in cultures

of their adult counterparts.

Myeloid potential was highest in the CD34+Lin-CD7-CD10- fractions, and

dramatically down regulated in CD7+ or CD10+ subsets, regardless of source. These

results are consistent with clonal assays conducted with methylcellulose cultures. With

two analyses of cord blood, most non-lymphoid progenitors were present in the CD7-

subset (710.8 ± 74.6 colonies / 1000 cells). However, it is noteworthy that an average

of 170.8 (± 50.8) colonies per 1000 cells were also obtained when the CD7+ fraction was

plated and 95% of them were erythroid. Similarly, the CD34+ Lin- CD7-CD10- fraction of

adult marrow contained most (an average of 60% in three experiments) of the

granulocyte/macrophage progenitors (CFU-G/M). As with cord blood cells, 98% of the

colonies obtained with the CD34+Lin-CD7+ fraction were erythroid (BFU-e). Insufficient

numbers of the rare CD7+CD10+ subset of adult marrow precluded their analysis in




18

these cultures.

Weekly examination revealed that myeloid progeny appeared quickly and then

declined in cultures initiated with the CD34+Lin-CD7-CD10- fraction of cord blood, while

myelopoiesis was more persistent in cultures of their adult counterparts (Figure 4B, C).

Progressive production of B and NK lineage cells was consistent with the primitive

nature of progenitors in this fraction. While transient B lineage differentiation was found

with the CD34+Lin-CD7+CD10- fraction, this was never the case with the comparable

adult marrow subset. The adult population also yielded a wave of NK lineage cells as

contrasted with sustained production from neonatal cells.

Neonatal or adult CD10+ fractions contained B lineage precursors that

differentiated within two weeks in culture and CD56+ NK lineage cells reached peak

values one week later. This analysis revealed large age related changes in the

potential of highly purified cord blood and adult marrow 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 described

above were also subjected to RT-PCR analysis (Figure 5). Transcripts associated with

B lymphocyte lineage differentiation (RAG-1, RAG-2, EBF and Pax5) were markedly up

regulated in the CD34+Lin-CD10+CD7- population relative to the more primitive CD34+

Lin-CD10-CD7- 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.




19

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 three 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.




20

Discussion

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. While 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 cord blood.

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 re-circulating 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




21

an exceptionally large fraction of small pre-B cells in that site are Ki-67+.45 As discussed

below, 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 utilized 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-CD24hiCD10+CD38+) B cells were scarce. The correlation between

percentages of naïve CD38+ B cells and cycling pre-B cells was not as good as the

relationship between pre-B cells and immature B cells (Figure 2). Cells with a naïve

phenotype might randomly leave marrow to enter the peripheral B cell pool, as has

been suggested for newly formed B cells in mice.53

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- cells have

been determined in other studies to be responsible for engraftment of NOD/SCID mice,




22

but uncharacterized accessory cells among the CD34- fraction of hematopoietic cell

suspensions can influence the outcome.41,42,51 To minimize this parameter, we added a

constant number of cord blood CD34- mononuclear cells to each of the transplant

samples, and human CD19+ cells were easily detectable in marrow of all transplanted

mice. As others have shown, stem cells within cord blood are exceptionally efficient for

engraftment of immunodeficient mice.48-50 We estimate that seven weeks after

transplantation there were 111 ± 98 human CD19+ cells present in each mouse femur

per injected CD34+CD38- cord blood cell. This value is 10 to 100 fold higher than we

obtained with precursors isolated from adult marrow.

Limiting dilution cultures initiated with highly purified CD34+CD38- cells provided

an independent means of assessing differentiation potential, and one that should be

less influenced by contaminating accessory cells. Cord blood precursors had a higher

cloning efficiency in vitro than adult marrow derived cells, and more of the responding

wells generated CD19+ lymphocytes (Table 2). This result is compatible with

observations made in another study, using a different culture system54 and suggests the

possibility of an intrinsic difference between neonatal versus adult progenitors.

Since 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




23

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. While flow cytometry revealed proportions of lymphocyte

precursors in most marrow specimens to be remarkably constant, functional precursor

frequencies among CD34+CD38- cells were highly variable (Table 2).

Flow cytometry experiments revealed that the composition and patterns of

surface marker expression differ substantially between cord blood and adult marrow.

The interesting CD34+CD38-CD7+CD10- subset recently described in cord blood33 has

no direct counterpart in adults. Furthermore, the cord blood CD34+CD38-CD7-CD10-

subset differed substantially from the comparable adult population with respect to

expression of CD45RA. Given these changes, we elected to sort three populations for

side-by-side comparisons in culture studies. CD10+CD19- cells comprise a very small

proportion (2.5±1.1%) of the 1% of marrow mononuclear cells that express CD34+ (30,32

and Figure 3). Most of these CD34+CD10+CD19- cells also express TdT30,32,61 and data

not shown) and already have D-JH rearrangements.34 Previous reports32,34 suggest that

this fraction is a rich source of early B lineage precursors. Our culture analyses

substantiate that conclusion and it is noteworthy that cord blood and adult progenitors

were equivalent in potential for B lineage differentiation (Figure 4). PCR analysis of

CD34+Lin-CD7-CD10+ cells from adult marrow revealed substantial expression of five

genes required for B lymphopoiesis (Figure 5). However, it is also important to stress




24

that CD10+ cells have some myeloid potential that can be appreciated in long term

cultures but not in clonal assays32 (Figure 4 and data not shown). Thus, a completely

lymphoid restricted progenitor has not been isolated from adult human marrow.

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-CD7+ fraction of cord blood contains what might be the human equivalent

of common lymphoid progenitors described in mice.12,33 We confirm that cord blood

CD34+CD7+CD10-CD19- cells can rapidly generate small numbers of CD19+ cells in

culture (Figure 4 and data not shown). Unlike the previous report, we detected some

non-lymphoid, and especially erythroid precursors in this population. This may be

because we did not additionally select for CD38- cells in cord blood. However, as noted

above, there are no CD38-CD7+ cells in adult human marrow and the total CD34+Lin-

CD7+CD10- fraction of marrow was a poor source of B lineage progenitors (Figures 3

and 4). PCR analysis of carefully sorted cells substantiated the culture results and

showed a considerable enrichment for cells expressing the CD122 receptor for IL-15/IL-

2 required for NK lineage differentiation (Figure 5). It was previously reported that the

CD34+CD7+ fraction of adult marrow contains precursors of NK cells.46 Thus, CD7

expression may characterize an intermediate stage between stem cells, erythroid cells

and pro-B cells in neonates, but be more closely associated with NK lineage restricted

precursors in adult marrow.

To summarize, several distinctive adult characteristics of B lymphocyte

precursors may be acquired during the neonatal or adolescent period. The primitive

CD34+Lin-CD10-CD7- fraction retains differentiation potential throughout life, but is much




25

less efficient than cord blood cells with the same phenotype. Cells bearing CD10+

include many early B lineage precursors and ones in neonates and adults appear to

have equivalent differentiation potential. All subsequent stages of B lineage

differentiation are represented in constant ratios in marrow and numbers of proliferating

pre-B cells closely correlate with numbers of newly formed B cells. The search must

continue for lymphoid restricted progenitors in human marrow and our understanding of

early differentiation events is incomplete.




26

Acknowledgments

P.W.K. holds the William H. and Rita Bell Chair in biomedical research.




27

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36

Table 1. Adult human bone marrow contains B lymphocyte precursors revealed

by transplantation of immunodeficient NOD/SCID mice.

Cells InjectedNo. of cells

Injected

No. of

CD34+CD38-

cells injecteda

No. of

engrafted/injected

mice (%)

Percentage of

hCD45+CD19+

cells in BM of

chimeras

Fetal BM CD34+ 2.9 – 3 X 106 28.5 – 30 x 103 5/6 (83%) 1.54 (± 1.28)

CB CD34+ 5 X 105 4 x 103 11/11 (100%) 1.02 (± 0.86)

Adult BM CD34+ 2 X 106 39.6 – 72 x 103 5/5 (100%) 0.22 (± 0.16)

CB CD34- 5 x 106 < 0.01 0/5 < 0.02

a Numbers of CD34+CD38- cells were determined by flow cytometry, using small

aliquots of each preparation of CD34+ enriched cells. These results were obtained in

two very similar, but independent experiments.




37

Table 2. Adult human bone marrow contains functional B lineage precursors that

can be enumerated in limiting dilution cultures of CD34+CD38- cells.

Source of CD34+CD38-

cells

Clonogenic Frequency Frequency of cells able to

give rise to CD19+ cells

Cord Blood 12.5a (11,11 – 14,29)b 6.67a (5.56 – 8.33)b

Adult BM 24 years 6.09 (4.84 – 13.53) 1.16 (0.77 – 1.96)

Adult BM 44 years 9.78 (7.78 – 19.01) 1.04 (0.67 – 1.79)

Adult BM 45 years 3.69 (2.78 – 6.56) 0.05 (<0.01 - 0.12)

Adult BM 70 years 4.5 (3.82 – 8.83) 0.18 (0.07 – 0.31)

Adult BM 88 years 3.6 (3.24 – 4.26) 0.15 (0.02 – 0.32)

a Percentage , b95% Confidence Interval (CI).




38

Figure Legends

Figure 1. Relative frequencies and mitotic activity of B lineage precursors in

bone marrow do not change during adult life. (A) The incidences of pro B cells

(CD34+CD10+CD19+), pre-B cells (CD19+ sIgM-cµ+), immature B (IgM+CD24hiCD10+IgD-

), naïve B (IgM+CD24hiIgD+CD38+/-) and mature B (IgM+CD24loIgD+CD38-) cells were

determined by flow cytometry and shown as percentages of total viable CD19+

lymphocytes (%) ± S.D. (B) Expression of the Ki-67 nuclear proliferation antigen was

determined by flow cytometry, using low angle light scatter to resolve large and small

lymphocytes. The data represent average percentages of pro B cells (CD34+CD19+),

and pre-B cells (CD19+sIgM-cµ+) ± S.D. Differences between groups were not

statistically significant (p>0,05).

Figure 2. Profiles of immature and naive B cells in some deficient marrow

specimens, reveals a correlation between cycling pre-B cells and immature B

cells. FACS profiles of immature (IgM+CD24hiCD38+IgD-) and naive

(IgM+CD24hiCD38+/-IgD+) B cells from a normal patient (A-B) are compared to a

deficient one (C-D). Immature B cells were also CD10+ (not shown). The index of

cycling pre-B cells in exceptional patient samples correlated with production of

Immature B cells (E), and less closely with naïve B cells (F).

Figure 3. Early lympho-hematopoietic progenitors in adult marrow differ from

ones in cord blood with respect to surface marker characteristics. (A) CD7/CD10

profiles are shown for CD34+ Lin- cells in cord blood and adult marrow. CD34+ Lin-




39

CD10- CD7- and CD34+ Lin- CD10- CD7+ cells were sorted from cord blood and adult

marrow, and stained with CD45RA-PE (B) or CD38-PE (C). These data are

representative of three independently sorted samples.

Figure 4. Early lympho-hematopoietic progenitors in cord blood and adult

marrow differ with respect to expansion and differentiation potential. CD34+Lin-

CD10-CD7-, CD34+Lin-CD10-CD7+, and CD34+Lin-CD10+CD7- cells were sorted from

cord blood or adult marrow, and cultured with MS-5 stromal cells (1 x 104 cells/flask in

each fraction). Yields of CD19+ B, CD56+ NK and CD13+/CD33+ myeloid lineage cells

in 3-week cultures are shown (A). The B, NK and myeloid lineage compositions were

also determined weekly in cultures initiated with fractions sorted from cord blood (B) or

adult marrow (C). Similar results were obtained in two independent side-by-side

comparisons between cord blood and adult marrow.

Figure 5. B lineage characteristics are established by the CD10+ stage in adult

marrow. Expression of lineage-related genes was examined in the CD34+Lin-CD10-

CD7- (closed bar), CD34+Lin-CD10-CD7+ (shaded bar) and CD34+Lin-CD10+CD7-

(dotted bar) fractions sorted from adult marrow. RT-PCR was used to amplify

transcripts for the indicated genes and the results were normalized according to

GAPDH expression.




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