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Originally published online as doi:10.1189/jlb.0706457 on August 25, 2008

Published online before print August 25, 2008
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(Journal of Leukocyte Biology. 2008;84:1557-1564.)
© 2008 by Society for Leukocyte Biology

Characterization of phenotypically distinct B-cell subsets and receptor-stimulated mitogen-activated protein kinase activation in human cord blood B cells

Yun Jung Ha*, Yeung-Chul Mun{dagger}, Chu-Myong Seong{dagger} and Jong Ran Lee*,{ddagger},1

* Division of Life and Pharmaceutical Sciences and Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul, Korea;
{dagger} Department of Hematology-Oncology, College of Medicine, Ewha Womans University, Seoul, Korea; and
{ddagger} Department of Life Science, College of Natural Sciences, Ewha Womans University, Seoul, Korea

1 Correspondence: Division of Life Science, College of Natural Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, Korea. E-mail: jrlee{at}ewha.ac.kr


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ABSTRACT
 
Human cord blood (CB) is a valuable source of hematopoietic stem cells, but clinical reports have indicated slow recovery of B-cell development and function after CB transplantation. To investigate the basis of these B-cell defects in reconstitution, we characterized B cells purified from CB. We compared B-cell receptor activation and B-cell subsets in CB, bone marrow (BM), and peripheral blood (PB). We found that in CB B cells activation of extracellular signal-regulated kinase (ERK) and p38 following ligation of CD40 but not of the B-cell antigen receptor (BCR) was inefficient. The patterns of expression of CD5, CD34, and CD40 in the B-cell population of CB were similar to those in PB rather than in BM. The B cells in CB contained an increased proportion of B cells expressing a high level of CD24 and a low proportion of B cells expressing CD27, pointing to the presence of circulating CD24high immature transitional and CD27– naive B cells. CD40-mediated activation of ERK and p38 was also minimal in these B cells of CB. These findings may account for the functional defects of B cells in transplanted CB.

Key Words: B-cell antigen receptor • CD40 • signal transduction • transitional B cells


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INTRODUCTION
 
Human cord blood (CB) is widely used as a source of hematopoietic stem cells, and transplantation of CB has many advantages over transplantation of bone marrow (BM) or peripheral blood (PB) [1 2 3 ]. Among these are relatively easy enrichment and availability and a low incidence of severe graft vs. host disease (GVHD) [1 2 3 ]. However, CB transplantation has been limited to children, because of the small volumes available. In addition, there have been clinical reports of slow recovery of lymphocyte development and function [4 5 6 ]. In particular, B-cell recovery after CB transplantation has been shown to be defective, with limited proliferation, differentiation, and Ig production both in vivo and in vitro [4 5 6 ]. Though IgM production was readily detectable, the production of IgG, IgA, and IgE was low [7 , 8 ]. As the interaction between CD40 on B cells and its ligand (CD40L) on activated CD4+ T cells delivers a key signal for Ig isotype switching [8 9 10 ], the defective B-cell responses could be the result of inefficient signaling via surface receptors.

To identify the defects in B cells recovered after CB transplantation, we studied characteristics of circulating B cells purified from CB. We investigated the surface receptor-mediated activation patterns of extracellular signal-regulated kinase (ERK) and p38, which control B-cell activation, as well as their death or survival [11 12 13 14 ]. We also analyzed the developmental status of circulating B cells. Comparison of the mononuclear cells (MC) and B cells in CB, BM, and PB revealed not only inefficient activation of ERK and p38 mitogen-activated protein (MAP) kinases after CD40 stimulation of CB B cells, but also an increased frequency of CD24highCD38highIgD+ B-cell subset that represents immature transitional B cells, as well as little frequency of CD24highCD38– memory B-cell population [15 16 17 18 19 20 21 22 23 24 ]. Further signaling studies performed on the different B-cell subsets also revealed inefficient activation of ERK and p38 after CD40 stimulation of CB B cells, more strikingly in transitional B-cell population.

Collectively, these results suggest that the reported defects in the developmental and functional recovery of B cells after CB transplantation are due to inefficient CD40 receptor-mediated signaling events, as well as the presence of a high proportion of immature transitional B cells and an insufficient memory B-cell population.


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MATERIALS AND METHODS
 
Antibodies (Abs) and reagents
For flow cytometry, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD5, CD24, CD27, CD34, CD40, IgD, R-phycoerythrin (R-PE)-conjugated mouse anti-human CD19, CD38, allophycocyanin (APC)-conjugated mouse anti-human CD19, and phycoerythrin-cyanin 5 (PE-Cy5)-conjugated mouse anti-human CD38, CD40 monoclonal Abs (mAbs) were purchased from BD PharMingen (San Diego, CA, USA). R-PE-conjugated anti-phospho-ERK1/2 (T202/Y204) and anit-phospho-p38 MAP kinase (T180/Y182) were from BD PhosFlow (San Diego, CA, USA), and FITC-, R-PE-, PE-Cy5-, APC-conjugated anti-mouse IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, CA, USA). For cell activation, goat anti-human IgM was from Southern Biotechnology Associates, Inc. (Birmingham, AL, USA), and mouse anti-human CD40 mAb (5C3) from BD PharMingen. For Western blot analysis, the rabbit polyclonal Abs against ERK2 and p38 were obtained from New England Biolabs Ltd. (Beverly, MA, USA), and Abs against the phosphorylated forms of ERK and p38 were also obtained from New England Biolabs Ltd. or from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal Abs against CD40, TRAF 2, 3, 5, and 6 were obtained from Santa Cruz Biotechnology. The secondary Ab, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, was from New England Biolabs or Bio-Rad (Hercules, CA, USA). B-cell isolation kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) was used for B-cell separation. Ficoll-Paque Plus and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech Co. (Arlington Heights, IL, USA), and D-sorbitol and phorbol 12, 13-myristic acetate (PMA) were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Samples and B-cell purification
Human umbilical CB samples were obtained from full-term newborns of healthy Korean mothers who delivered naturally. Blood was recovered with informed consent from the mothers. Samples of BM were obtained from healthy adult donors, who were normal on physical examination and routine hematological and biochemical screening. Samples of PB were collected from normal healthy donors. Written informed consent was obtained from all donors, and all studies were approved by the institutional human ethics review board of Ewha Woman’s University Medical Center. Samples of MC were isolated from CB, BM, and PB by Ficoll-Paque Plus density centrifugation. For stimulation, they were washed three times, and for isolation of the B-cell fraction, they were washed and suspended in phosphate-buffered saline (PBS; pH 7.4) containing 0.1% bovine serum albumin (BSA). The B-cell fraction was isolated by depletion of non-B cells with superparamagnetic microbead selection using a cocktail of mAbs and mini-Macs columns (Miltenyi Biotech). The efficiency of purification was verified by flow cytometry, counterstaining with R-PE-anti-human CD19 mAb, and normally reached in the range of 94–99%.

Detection of MAP kinase activation and Western blot analysis
Activation of the MAP kinases ERK and p38 following receptor ligation was assessed by measuring phosphorylation of the enzymes. Samples of MC (5x106) or B cells (2x106) isolated from CB, BM, and PB were incubated in medium alone or in medium containing anti-IgM (10 µg/ml), anti-CD40 (10 µg/ml), PMA (20 µg/ml), or sorbitol (0.6 M) at 37°C for various periods of time. The cells were lysed in 1% Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, as described previously [13 ]. Cell lysates were mixed with 2x Laemmli sample buffer, boiled, and subjected to SDS-10% PAGE. After transferring proteins to a nitrocellulose membrane, Western blot analysis was performed by blocking the membrane with 5% nonfat dried milk and incubating with Abs against the phosphorylated or unphosphorylated form of enzyme, followed by HRP-conjugated secondary Ab; for detection, we used ECL reagents. Phosphorylated enzyme was quantified by scanning the bands in phosphorylated and unphosphorylated Western blots and determining their intensities with Image/Gel Plotting software. Western blots for CD40, TRAF 2, 3, 4, and 6 proteins were performed with 25-30 µg of the lysates of isolated B cells from CB, BM, and PB.

Flow cytometry
The MC (1x106) from CB, BM, and PB were blocked with 5% BSA followed by direct 2, 3, or 4-color surface staining with various mAb combinations for 30 min on ice in staining buffer containing 1% BSA in PBS. After being incubated in medium alone or in medium containing anti-IgM (10 µg/ml) or anti-CD40 (10 µg/ml) for various times at 37°C, the MC (2x106) isolated from CB, BM, and PB were stained directly for surface molecules and fixed in 2% paraformaldehyde, permeabilized in ice-cold 95% methanol, followed by staining phosphorylated ERK or p38 MAP kinases. Control staining was performed by incubating the cells with FITC-, R-PE-, and PE-Cy5-conjugated anti-mouse IgG. Stained cells were washed and resuspended in HEPES buffer. Data were collected immediately using a 2, 3, or 4-color FACSCalibur and analyzed using CellQuest software (BD Biosciences, Mountain View, CA, USA). Ten thousand events were acquired for each sample. Fluorescence signals were analyzed as dot plots of the fluorescence intensity channels.


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RESULTS
 
Activation of ERK and p38 MAP kinases after ligation of surface IgM or CD40 in the MC from CB, BM, and PB
BCR and CD40 are important surface receptors involved in the activation and maturation of B cells during the humoral immune response, as well as in the development of plasma or memory cells [8 9 10 ]. As ERK and p38 MAP kinases play critical roles in B-cell activation and are also activated after ligation of these receptors [11 12 13 14 ], we investigated the activation of these kinases following stimulation with anti-IgM or anti-CD40 Ab in the MC isolated from CB, BM, and PB. Kinase activation was determined from the fraction of the phosphorylated form of the enzyme after stimulation of each receptor and was compared (by fold induction) to the response in the absence of receptor ligation for each time point (Fig. 1 ). The normalized values (phosphorylated form/total enzyme) were calculated by quantitating the intensity of the bands on Western blots of each enzyme.


Figure 1
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  		Figure 1. Activation of ERK and p38 MAP kinases after CD40 ligation is inefficient in the MC derived from CB. MC (5x106) isolated from CB (A and D), BM (B and E), or PB (C and F) were stimulated with medium containing control Ig (control), 10 µg/ml anti-IgM, or 10 µg/ml anti-CD40 for various times. The cells were also incubated with 20 µg/ml of PMA for 5 min and with 0.6 M D-sorbitol for 30 min as positive controls for ERK and p38 activation, respectively. Cell lysates were subjected to SDS-PAGE and phospho-ERK (P-ERK) or phospho-p38 (P-p38) Western blot analysis. The same blot was stripped and reprobed with anti-ERK2 Ab or anti-p38 Ab to ensure equal loading of the cell lysates. Representative Western blots are shown for MC from CB (n=5), BM (n=2), and PB (n=4). (A–C) The intensities of the bands of P-ERK and ERK2 were quantitated and normalized values (P-ERK2/ERK2) were calculated. From the normalized values the fold-ERK activation was determined for stimulation with anti-IgM and anti-CD40 relative to those for the control. The data shown in graphs are the averages ± SE of the normalized values from three separate Western blots for MC from CB and PB, and the averages + range of the normalized values from two separate Western blots for BM. (D–F) Similarly, fold inductions were calculated from the averages of the normalized values (P-p38/p38) from three separate Western blots for the MC from CB and PB, and from two separate Western blots for MC from BM.

When the averaged normalized values for enzyme activation in independent experiments were compared, stimulation with anti-IgM was seen to activate ERK and p38 within 5 min in the MC of all blood samples (Fig. 1) . The patterns of kinase activation differed among samples: 1) both ERK 1 and 2 were activated in the MC of BM and PB, but only ERK2 was activated in the CBMC as shown in Fig. 1 A-C ; 2) strong activation was followed by a rapid reduction within 30 min in the BMMC, whereas activation was relatively long-lasting in the MC from CB and PB (Fig. 1) . In contrast with the response to BCR ligation, the CBMC responded inefficiently to CD40 stimulation: slow and weak activation of the kinases is evident in Fig. 1 A and D . As in the case of the BMMC stimulation with anti-IgM, strong activation followed by a reduction within 30 min was induced by CD40 stimulation (Fig. 1 B and E) , although ERK activation in the BMMC after CD40 stimulation was weaker than after BCR ligation. However, the BCR- and CD40-induced activation of ERK and p38 MAP kinases in the PBMC was relatively long-lasting, as shown in Fig. 1 C and F .

Signaling molecule expression, ERK and p38 activation via surface receptor, IgM or CD40, in B cells isolated from CB, BM, and PB
As there are potentially other cell types that share receptors with the B-cell population, we examined the activation of ERK and p38 after ligation of surface IgM or CD40 in B cells purified by depletion of non-B cells from CB and PB (Fig. 2 A-D ). As in the response of the MC, stimulation with anti-IgM Ab resulted in rapid and strong activation of ERK and p38 MAP kinases in the B cells of CB and PB samples. Stimulation with anti-CD40 also induced strong activation of these kinases in the B cells from PB (Fig. 2 B and D) . In contrast with the response to BCR ligation, the B cells from CB responded inefficiently to CD40 stimulation as in the response of the CBMC (Fig. 2 A and C) .


Figure 2
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  		Figure 2. Inefficient activation of ERK and p38 MAP kinases after CD40 ligation in the B cells derived from CB is not due to defective expression of the CD40 receptor or TRAF molecules. B cells (2x106) isolated from the MC of CB (A and C) or PB (B and D) were stimulated as in Fig. 1 , and lysates were subjected to SDS-PAGE and Western blot analysis for P-ERK (A and B) and P-p38 (C and D). The same blot was stripped and reprobed with anti-ERK2 Ab (A and B) or with anti-p38 Ab (C and D) to ensure equal loading. Results are representative of three CB samples and two PB samples. Fold inductions were calculated from the averages of the normalized values (P-ERK/ERK, P-p38/p38). The data shown in graphs are the averages ±SE from three separate Western blots for CB B cells, and the averages + range from two separate Western blots for PB B cells. (E) Lysates (25 µg) of CD19+ B cells isolated from BM, CB, and PB were subjected to SDS-PAGE and Western blot analysis for CD40 and TRAF2, 3, 5, 6. Results are representative of two BM aspirates and three samples of CB and PB.

Western blot analysis of lysates demonstrated similar expression of CD40 in the B cells of all blood samples, as shown in Fig. 2E . The expression of the adaptor molecules, tumor necrosis factor receptor-associated factor (TRAF) 2, 3, 5, and 6, which are known to play important signaling roles in the pathway downstream of CD40 [25 26 27 ], was also detected with similar levels (Fig. 2E) . The levels of the CD40 and TRAF mRNAs were also similar in all of the B cells from BM, CB, and PB (data not shown). These results suggest that the inefficient activation of ERK and p38 MAP kinases by stimulation of CD40 in the isolated B cells and the MC of CB is not due to any immediate signaling defect associated with CD40 receptor ligation.

Phenotypic characteristics of the CD19+ cells from the CBMC, BMMC, and PBMC
As the patterns of activation of MAP kinases following BCR and CD40 ligation differ, we next investigated a possibility of phenotypic differences of the CD19+ cells derived from the MC of CB, BM, and PB (Fig. 3 ). It is known that the expression of CD5 indicates a difference in phenotype or development (fetal/adult; Type-I/Type-II; B1/B2) [28 29 30 ] and that the expression of the costimulatory molecule CD40 accounts for T-dependent Ab responses [8 9 10 ]. We first analyzed the surface expression of CD40 and CD5 and found that most of the CD19+ B cells from CB and PB were CD40+ as shown in Fig. 3 Aa and Ca , while the ratio of CD40–/CD40+ was 1/2.6 in the BM CD19+ cells shown in Fig. 3Ba . An analysis of CD5 expression showed a more than threefold higher proportion of CD5+ cells in the CD19+ CB and PB cells than in the CD19+ BM cells (Fig. 3 A-Cb) .


Figure 3
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  		Figure 3. Surface molecule expression patterns of CD19+ cells from CBMC, BMMC, and PBMC. The MC purified from CB (A), BM (B), and PB (C) were doubly labeled, and standard flow cytometric analyses were performed. Double staining was performed with mAb specific for the B-lineage marker CD19 (PE) and each FITC-conjugated mAbs specific for CD40 (a), CD5 (b), CD34 (c), CD24 (d), or CD27 (e). The level of CD24+ was classified into high, medium, and low using a dashed line as seen in each panel. Results are representative of data from at least three CB, BM, and PB samples.

We next compared the maturity and differentiation of the CD19+ cells in the B-cell populations isolated from the CBMC, BMMC, and PBMC. To do this, we used a set of surface markers, CD34 for pro-B cells, CD24 for both immature and mature B cells, and CD27 for memory B cells [15 16 17 18 19 20 21 22 23 24 , 31 32 33 ]. Our analysis demonstrated that the majority of the CD19+ B-cell population of CB, BM, and PB were in the stage of CD34– and were immature and naïve B cells (CD24+ and CD27–), although subtle differences were noticed among the B cells from the different sources, as shown in Fig. 3 A-Cc-e .

Compared with less than 2% of the CD19+ CB and PB cells (Fig. 3 Ac and Cc) , 8.3% of the CD19+ BM cells were CD34+ (Fig. 3Bc) . The fraction of CD27+ cells among the CD19+ CB cells (Fig. 3Ae , ~7%) was much lower than that in the CD19+ BM and PB cells (Fig. 3 Be: ~23% and Ce: ~28%) . Above all, comparison of CD24 expression, which correlates inversely with the degree of B-cell maturity, indicated that all the CD19+ CB cells were CD24+, medium~high as shown in Fig. 3Ad , whereas the majority of the CD19+ cells in the BM and PB were CD24+, low~medium (Fig. 3 Bd and Cd) . In particular, none of the CD19+ PB cells was CD24+, high (Fig. 3Cd) , although the CD19+ BM cells were CD24+, low~high (Fig. 3Bd) . These results demonstrate that a large proportion of the CD19+ CB cells were at an early stage of differentiation and maturity.

Characterization of B-cell subsets from CB, BM, and PB
As a slightly different complement of immature, mature, and memory B-cell population was shown in each sample of CB, BM, and PB as shown in Fig. 3 , further analyses were performed to compare the maturity and differentiation of the B-cell populations from CB, BM, and PB. To identify the phenotype and frequency of B-cell subsets, combinations of developmentally regulated surface markers such as CD24 vs. CD38 and IgD vs. CD38 in combination with the B-lineage marker CD19 were used (Fig. 4 ).


Figure 4
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  		Figure 4. Phenotype and frequency of B-cell subsets from CB, BM, and PB by the surface markers for maturity and differentiation. (A–C) The MC purified from CB (A), BM (B), and PB (C) were labeled by direct 4-color surface staining with mAbs specific for the B-lineage marker CD19 (APC), CD24 (FITC), CD38 (PE), CD40 (PE-Cy5), and standard flow cytometric analyses were performed. (a) On the basis of the relative expression of CD24 and CD38, three populations of B cells (R1; CD24highCD38high, R2; CD24+CD38+, R3; CD24highCD38–) were compared among CB, BM, and PB. (b) The level of CD40 expression was compared in each population by the CD38 staining. Results are representative of data from at least three CB, BM, and PB samples. (D–F) The MC purified from CB (D), BM (E), and PB (F) were labeled by direct 3-color surface staining with mAbs specific for the B-lineage marker CD19 (PE), IgD (FITC), CD38 (PE-Cy5), and standard flow cytometric analyses were performed. Immature populations (CD38high) were compared among CB, BM, and PB by the IgD/CD38 staining: IgD–CD38high pro/pre B cells (dashed gate) and IgD+CD38high transitional B cells (solid gate). Results are representative of data from at least three CB, BM, and PB samples.

As the level of CD24 expression differed among B cells from CB, BM, and PB as shown in Fig. 3 , B-cell subsets by the CD38/CD24 staining were compared. All three immature transitional (R1: CD24highCD38high), mature naïve (R2: CD24+CD38+), and memory (R3: CD24highCD38–) B-cell subsets were identified in BM and PB, although differences in the proportion of each subset were noticed (Fig. 4 Ba and Ca) . In contrast, transitional and naïve B-cell subsets, but little memory subset was identified in CB (Fig. 4 Aa) . The surface CD40 expression was also compared in each B-cell subset from CB, BM, and PB. From all three blood sources similar level of the CD40 surface expression was found in B-cell subsets by CD38 staining (Fig. 4 A-Cb) .

The phenotype of immature transitional B-cell subset was further compared by the CD38/IgD staining. Three B-cell subsets were identified: pro-pre (IgD–CD38high), mature naïve (IgD+CD38+), and immature transitional (IgD+CD38high). The proportion of CD38high B-cell population was higher in CB and BM than in PB (Fig. 4 D-F) . The majority of CD38high population was pro/pre and transitional B cells in BM and CB, respectively (Fig. 4 D and E) . In both CB and PB, a majority of B cells were mature naïve population (Fig. 4 D and F) .

Activation of ERK and p38 MAP kinases after the ligation of surface IgM or CD40 in the phenotypically distinct B-cell subsets of CB, BM, and PB
As we and others have demonstrated the phenotypic differences of B-cell subsets derived from CB, BM, and PB, as well as insufficient memory B-cell subsets in CB [18 , 31 , 34 ], we investigated further whether the inefficient activation of ERK and p38 MAP kinases by CD40 stimulation in the B cells from CB is due to intrinsic signaling defects of B cells or relatively little memory B-cell population in CB. We examined the activation of ERK and p38 after the stimulation with anti-IgM or anti-CD40 Ab in the B-cell subsets that were identified by the CD38/CD24 staining. The activation of these kinases was compared among immature transitional and mature naïve B-cell subsets of CB, BM, and PB, as well as memory B-cell subsets of BM and PB, respectively.

Mean fluorescence intensities of the phosphorylated ERK or p38 MAP kinases by direct intracellular staining were compared in each B-cell subset of three blood sources. As the activation of ERK or p38 kinase after the stimulation with anti-IgM or anti-CD40 varies enormously among individual blood samples, the activation of these kinases in each B-cell subset was analyzed by the percentage of maximum response induced by stimulation for various times. Stimulation with anti-IgM induced strong activation of ERK (a) and p38 (b) kinases in the immature transitional (A–C) and mature naïve (D–F) B-cell subsets of three blood sources, although a relative degree of the activation differs in various samples (Fig. 5 ). Stimulation with anti-CD40 also induced strong activation of these kinases in these B-cell subsets of BM and PB (Fig. 5 B, C, E, and F) . In contrast to the response with anti-IgM, B cells in transitional and mature naïve subsets of CB responded inefficiently to CD40 stimulation (Fig. 5 A and D) . Strong activation of ERK and p38 kinase was also induced in memory B-cell subsets of BM and PB after the stimulation with anti-IgM or anti-CD40 (data not shown). These results suggest that the inefficient activation of ERK and p38 MAP kinases by CD40 stimulation in the B cells from CB is due to hyporesponses of B cells to the CD40 ligation, as well as insufficient memory B-cell subsets in CB.


Figure 5
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  		Figure 5. Activation of ERK and p38 MAPK after the ligation of IgM or CD40 in B-cell subsets from CB, BM, and PB. The MC purified from CB, BM, and PB were incubated in medium alone or in medium containing anti-IgM (10 µg/ml) or anti-CD40 (10 µg/ml) for indicated times at 37°C. These cells were immediately stained with mAbs specific for CD24 (FITC) and CD38 (PE-Cy5) before fixing and intracellular staining with R-PE-conjugated mAb for P-ERK, or P-p38. Standard flow cytometric analyses were performed. Activation of ERK (A–C) and p38 (D–F) was compared in each B-cell population (a; CD24highCD38high, b; CD24+CD38+) among CB (A and D), BM (B and E), and PB (C and F). From the mean fluorescence intensities of P-ERK and P-p38, at each time point, the activation was calculated as the % of maximum (max) response after the stimulation with anti-IgM or anti-CD40 in each B-cell population. The symbols in graphs represent individual samples of each B-cell source.


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DISCUSSION
 
The clinical use of umbilical CB as a source of stem cells for BM reconstitution has been characterized by an apparently low incidence of severe GVHD compared with adult BM [1 2 3 ]. Thus, CB may overcome some of the limitations of sibling and unrelated BM transplantation, and there is increasing clinical interest worldwide in CB transplantation as an alternative therapeutic protocol for BM reconstitution. Understanding the recovery of lymphocyte development and function after CB transplantation is much needed at this point.

Studies on the maturity and differentiation of the MC are important in this context. The immunophenotypic features of CB and PB samples have been analyzed by flow cytometry using a large panel of mAb, and significant difference between CB and adult PB lymphocytes were revealed; in particular, the immaturity of the CB cells [35 36 37 ]. The reduced CB lymphocyte alloreactivity in allogeneic transplantation is explained by the majority of naïve T lymphocytes. The severe immunodeficiency in the recipient humoral immune defense after CB transplantation also results from the fact that most of the B1 cells have the newborn B-cell repertoire and are capable of producing polyreactive IgM and natural autoantibodies but not IgG.

Although the phenotypic characterization of the CBMC is consistent with the previously reported advantages and limitations of the use of CB for BM replacement, further studies are required in order to understand the precise mechanisms involved. Several studies have focused on neonatal CB-derived T cells and suggested possible molecular mechanisms underlying the immature immune response of CB T cells: 1) inadequate phospholipase C{gamma} activation associated with very low expression of the Src kinase, Lck [38 ], 2) selective deficiency in protein kinase C (PKC) isoenzyme expression followed by inadequate PKC-MAP kinase signaling [39 ], and 3) blockage of CD3- and CD28-mediated signaling events [40 ]. These signaling molecule defects and the signaling immaturity may be responsible for the reduced lymphokine production and dysfunction of FasL-mediated cytotoxicity and may account also for the low incidence of severe GVHD.

In this study, we examined CB B-cell development and function, with a special focus on the mechanisms and molecules that regulate the expression of humoral immunity. Defects in signaling via CD40 in the CB B cells were demonstrated by assaying MAP kinase activation (Figs. 1 , 2 , and 5) . The CD40 expression level and the ratio of B1/B2 cells among the B cells from the three different sources (Figs. 3 and 4) , as well as the expression of molecules involved in CD40 signaling (some data not shown; adaptor TRAF molecules shown in Fig. 2E ) were not sufficient to explain the defective MAP kinase activation after ligation of CD40. Comparison of the maturity and differentiation of the cells using surface markers for pro/pre, immature, mature, and memory B-cell populations, however, distinguished the CD24+, medium~high CB B cells from the majority CD24+, low~medium BM and PB B cells (Fig. 3) .

Human transitional B cells have been characterized not only in terms of phenotype, but also function [22 23 24 ]. Their existence is suggested by the recent demonstration of a population of PB cells distinguishable from mature B cells on the basis of their CD24highCD38high phenotype [15 16 17 18 ]. The CD24+, medium~high B cells of the CB demonstrated in our study could all be in a transitional B-cell stage and may not mature further. Increased level of CD24highCD38highIgD+ immature transitional B-cell subsets, as well as little frequency of CD24highCD38– memory B-cell population was, in fact, characterized for CB B cells (Fig. 4) .

Further signaling studies performed on the different B-cell subsets revealed inefficient activation of ERK and p38 after CD40 stimulation of CB B cells, more strikingly in transitional B-cell population. Thus, the reported defects in the developmental and functional recovery of B cells after CB transplantation are due to inefficient CD40 receptor-mediated signaling events, as well as the presence of a high proportion of immature transitional B cells and an insufficient memory B-cell population.

The immature immune response and low incidence of severe GVHD in CB transplantation were shown to be due to attenuation of T cell activation signaling [41 ]. Similarly, there may be some distinct characteristics of the physical linkage of signaling molecules, possibly involving lipid rafts that may affect activation of CB B cells. However, the exact basis of the inefficient CD40 signaling in these CB B-cell types remains to be determined.


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ACKNOWLEDGEMENTS
 
This study was supported by a grant (02-PJ1-PG3-21202-0004) from the Korea Health 21 R&D Project, Ministry of Health and Welfare, a grant (R04-2004-000-10077-0) from the Korea Research Foundation, another grant (R01-2006-000-10429-0) from the Basic Research Program of the Korea Science and Engineering Foundation (KOSEF), and the National Core Research Center program (R15-2006-020) of the Ministry of Education Science, and Technology and the KOSEF through the Center for Cell Signaling and Drug Discovery Research at Ewha Womans University. We thank members of our laboratory for willingly providing PB samples, and the donors who consented to give CB and BM samples. Y. J. Ha was supported in part by the Brain Korea 21 Program from the Korea Ministry of Education.

Received July 20, 2006; revised August 4, 2008; accepted August 6, 2008.


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