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Increased Expression of CD27 on Activated
Human Memory B Cells Correlates with
Their Commitment to the Plasma Cell
Lineage
Danielle T. Avery, Julia I. Ellyard, Fabienne Mackay, Lynn
M. Corcoran, Philip D. Hodgkin and Stuart G. Tangye
J Immunol 2005; 174:4034-4042; ;
doi: 10.4049/jimmunol.174.7.4034
http://www.jimmunol.org/content/174/7/4034
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References
The Journal of Immunology
Increased Expression of CD27 on Activated Human Memory
B Cells Correlates with Their Commitment to the Plasma
Cell Lineage1
Danielle T. Avery,* Julia I. Ellyard,*† Fabienne Mackay,‡ Lynn M. Corcoran,§
Philip D. Hodgkin,§ and Stuart G. Tangye2*
O
ne of the primary functions of mature B cells is the production of protective high affinity Ig following terminal
differentiation into plasma cells (PC).3 In humans, PC
can be identified by the up-regulation in expression of CD38 and
the concomitant decrease in CD20 (CD38⫹⫹CD20⫾) (1, 2). Cells
with this phenotype have been detected in lymphoid tissues such as
spleen (3), tonsils (4, 5), intestine (6, 7), bone marrow (BM), and
peripheral blood (1, 5). Importantly, PC are overrepresented not
only in malignancies (2, 8), but also autoimmune disorders such as
systemic lupus erythematosus (9, 10). Therefore, delineating the
mechanisms involved in generating these cells is necessary for a
complete understanding of the humoral immune response as well
as PC dyscrasias.
Ig-secreting cells (ISC) resembling PC can also be generated in
vitro by culturing human germinal center or memory B cells with
*Centenary Institute of Cancer Medicine and Cell Biology, and †University of Sydney, Sydney, New South Wales, Australia; ‡Garvan Institute of Medical Research,
Darlinghurst, New South Wales, Australia; and §Walter and Eliza Hall Institute of
Medical Research, Royal Melbourne Hospital, Victoria, Australia
Received for publication October 25, 2004. Accepted for publication January
19, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the National Health and Medical Research Council
(NHMRC) of Australia. S.G.T. is the recipient of an RD Wright Biomedical Career
Development Award from the NHMRC; P.D.H. is a Principal Research Fellow of the
NHMRC; L.M.C. is a Senior Research Fellow of the NHMRC.
2
Address correspondence and reprint requests to Dr. Stuart Tangye, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown 2042, New
South Wales, Australia. E-mail address: [email protected]
3
Abbreviations used in this paper: PC, plasma cell; BM, bone marrow; ISC, Igsecreting cell; SA, streptavidin; BAFF, B-cell activating factor belonging to the TNF
family; BAFF-R, BAFF receptor; BCMA, B cell maturation Ag; TACI, transmembrane activator of and CAML interactor; XBP-1, X box-binding protein-1.
Copyright © 2005 by The American Association of Immunologists, Inc.
BM stromal cells and/or activated T cells (11, 12), a follicular
dendritic-like cell line plus CD40L, IL-2, IL-4, and IL-10 (13, 14)
or the T cell-derived stimuli CD40L, IL-2, and IL-10 (15–17).
These cells have typically been identified by the increased expression of CD38, and the acquired ability to secrete high levels of Ig
(11–17). Such cells generated in vitro are rapidly proliferating (17–
20), suggesting they are more akin to plasmablasts detected in vivo
(21, 22), rather than mitotically inactive PC (23). By tracking the
proliferation history of activated B cells, we have recently found
that the differentiation of memory B cells into CD38-expressing
cells is IL-10 dependent, and increases in frequency with cell division; that is, CD38⫹ B cells appear in cultures of memory B cells
stimulated with CD40L and IL-10 once the cells have undergone
three or more rounds of cell division (17, 20, 24). Furthermore, the
survival requirements of the CD38⫹ B cells differed from CD38⫺
B cells present in the same culture inasmuch that CD38⫹ B cells
became independent of CD40L, and responsive to B-cell activating
factor belonging to the TNF family (BAFF), while CD38⫺ B cells
required continual stimulation with CD40L for their persistence
(11, 17, 25). However, we also found that only ⬃40% of CD38⫹
cells secreted Ig, and that there was a population of CD38⫺ ISC
(17, 25). Thus, the “PC” phenotype of CD38⫹ cells generated in
vitro did not correlate with PC function. To address this discrepancy, we have now examined in greater detail cultures of activated
memory B cells in an attempt to accurately identify the phenotype
of ISC generated in vitro. We found that CD27 is up-regulated on
the surface of memory B cells in an IL-10-dependent and divisiondependent manner, and that ISC segregated into the CD27high subset of activated memory B cells irrespective of the acquired expression of CD38. The CD27high ISC exhibited increased
expression of the transcription factors Blimp-1 and X box-binding
protein-1 (XBP-1), and reduced expression of Pax-5, as well as
phenotypic and migratory characteristics similar to those of human
0022-1767/05/$02.00
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Plasma cells (PC) or Ig-secreting cells (ISC) are terminally differentiated B cells responsible for the production of protective Ig.
ISC can be generated in vitro by culturing human B cells with the T cell-derived stimuli CD40L, IL-2, and IL-10. ISC have
traditionally been identified by the increased expression of CD38, analogous to primary human PC, and the acquired ability to
secrete Ig. By tracking the proliferation history of activated B cells, we previously reported that the differentiation of memory B
cells into CD38ⴙ B cells is IL-10 dependent, and increases in frequency with cell division. However, <50% of CD38ⴙ cells secreted
Ig, and there was a population of CD38ⴚ ISC. Thus, the PC phenotype of CD38ⴙ cells generated in vitro did not correlate with
PC function. To address this, we have examined cultures of activated memory B cells to accurately identify the phenotype of ISC
generated in vitro. We found that CD27 is also up-regulated on memory B cells in an IL-10-dependent and division-dependent
manner, and that ISC segregated into the CD27high subset of activated memory B cells irrespective of the acquired expression of
CD38. The ISC generated in these cultures expressed elevated levels of the transcription factors Blimp-1 and X box-binding
protein-1 and reduced levels of Pax-5, and exhibited selective migration toward CXCL12, similar to primary PC. We propose that
the differentiation of memory B cells into PC involves a transitional stage characterized by a CD27highCD38ⴚ phenotype with the
acquired ability to secrete high levels of Ig. The Journal of Immunology, 2005, 174: 4034 – 4042.
The Journal of Immunology
4035
primary PC. In contrast, nondifferentiated B cell blasts (i.e.,
CD38⫺CD27⫹) present in the same culture retained features of
memory B cells. These results reveal the dynamic ability of human
memory B cells to generate multiple cell fates following activation
with T cell-dependent stimuli to yield effector cells, namely ISC,
as well as sufficiently expand the pool of nondifferentiated memory
B cells to increase the size of this population of effector cells that
can then be rapidly activated following re-exposure to the
immunizing Ag.
At the completion of the primary or secondary cultures, a known number of CaliBRITE beads (BD Biosciences) were added to culture wells
before harvesting, and the number of viable B cells was calculated as a
function of the ratio of beads to live cells (29). All cultures were performed
using RPMI 1640 containing L-glutamine (Invitrogen Life Technologies),
10% FCS (CSL), 10 mM HEPES (pH 7.4) (Sigma-Aldrich), 0.1 mM nonessential amino acid solution (Sigma-Aldrich), 1 mM sodium pyruvate
(Sigma-Aldrich), 100 U/ml penicillin, 100 ␮g/ml streptomycin (SigmaAldrich), 100 ␮g/ml Normocin (InVivoGen), and 40 ␮g/ml apo-transferrin
(Sigma-Aldrich) and were conducted at 37°C in a humidified atmosphere
containing 5% CO2.
Materials and Methods
Immunofluorescent staining
Reagents
For phenotypic analysis, cells were incubated on ice with PE-, allophycocyanin-, or biotinylated specific mAb, or the appropriate isotype control,
followed by SA-PerCp and analyzed on a FACSCalibur using CellQuest
software (BD Biosciences). Binding of BAFF to activated B cells, as well
as assessment of expression of the different BAFF receptors (i.e., BAFF-R,
TACF, and BCMA) were determined as described previously (25)
Generation of CD70 transfectants
A complementary DNA encoding human CD70 was amplified from RNA
prepared from the CD70-expressing human B cell line JY by PCR using
Pfu polymerase and the following primers (Sigma-Genosys): 5⬘-GCA TGC
GGA TCC TTC CTT CCT TCT CGG CAG CG (BamH1 site underlined),
and 3⬘-GCA TGC GCG GCC GCA ATC AGC AGC AGT GGT CAG GG
(NotI site underlined). The resulting product was digested with BamH1 and
NotI, and ligated into pcdef3, a derivative of pEF-BOS containing the
neomycin-resistance gene (27). The mouse mastocytoma cell line P815
was transfected with CD70/pcdef3 by electroporation, and positive cells
were selected initially in the presence of G418 (Bio-Rad), and subsequently
by cell sorting.
Cells
Total human B cells (⬎98% CD19⫹) were isolated as previously described
(17, 28). Memory B cells were isolated by sorting on a FACStarPLUS or
FACSVantage (BD Biosciences) following labeling with FITC-anti-CD20
and PE-anti-CD27 mAb and collecting CD27⫹CD20⫹ B cells (17, 26).
CFSE labeling and B cell cultures
Primary cultures. Memory B cells were labeled with CFSE (29) and cultured in 48-well plates (4 ⫻ 105/ml; BD Labware) for 5 days with CD40L
alone (at a predetermined optimal dilution of the membrane preparation;
1/250), or in the presence of IL-10 (100 U/ml), or IL-2 (50 U/ml) plus
IL-10. The cells were harvested and expression of CD27 and CD38 was
then determined by immunofluorescence and flow cytometric analysis.
Secondary cultures. CFSE-labeled memory B cells were cultured for 4
days with CD40L, IL-2, and IL-10. The cells were harvested, washed, and
recultured (⬃2 ⫻ 105/ml) with IL-2 and IL-10 in the absence or presence
of CD40L (1/500 dilution) or BAFF (2.5 ␮g/ml) for an additional 4 days
(15, 17, 25). In some experiments, the activated memory B cells were
recultured with parental P815 or CD70/P815 cells in the presence of IL-2
and IL-10. Before culture, the P815 cells were fixed in 1% formaldehyde,
prepared in PBS, for 20 min (30), washed three times with PBS, and cultured in medium for 1 h to remove the formaldehyde. The memory B cells
and P815 cells were cultured at a ratio of 5:1.
Analysis of Ig secretion
ELISPOT. CFSE-labeled memory B cells were cultured with CD40L, IL-2,
and IL-10 for 4 days and then washed and recultured under the same conditions for a further 4 days. The cells were harvested and labeled with PE-antiCD27 mAb and allophycocyanin-anti-CD38 mAb. Different populations of
activated B cells, defined by division history and surface phenotype, were
sorted directly into ELISPOT plates (Multiscreen-HA plates; Millipore), and
the frequency of cells secreting IgM, IgG, and IgA was determined (17).
ELISA. CFSE-labeled memory B cells were cultured for 5 days with
CD40L, IL-2, and IL-10 and then labeled with PE-anti-CD27 and allophycocyanin-anti-CD38 mAbs. Different populations of activated B cells were
isolated by cell sorting and then recultured (⬃105 cells/500 ␮l/well) for a
further 2 days with CD40L, IL-2, and IL-10, after which time supernatants
were collected and the level of secreted Ig was determined (17).
Analysis of expression of transcription factors
Semiquantitative PCR was used to examine gene expression in different
populations of activated human memory B cells. B cell subsets were isolated by cell sorting, total RNA was extracted (Qiagen RNeasy Kit; Qiagen) and then transcribed into cDNA using oligo-dT (Boehringer Mannheim) or random hexamers (Invitrogen Life Technologies) as primer and
Superscript II RNase H⫺ reverse transcriptase (Invitrogen Life Technologies). Resulting cDNA was then normalized for expression of the constitutively expressed housekeeping gene GAPDH (5⬘-CCA CCC ATG GCA
AAT TCC ATG GCA, 3⬘-TCT AGA CGG CAG GTC AGG TCC ACC)
and then used as a template for PCR using REDTaq (Sigma-Aldrich) (3).
The following primers were used (Sigma-Genosys): Pax-5 5⬘-GCA TAG
TGT CCA CTG GCT CC; Pax-5 3⬘-CCA GGA GTC GTT GTA CGA GG;
BLIMP-1 5⬘-GAT GCG GAT ATG ACT CTG TGG; BLIMP-1 3⬘-CTC
GGT TGC TTT AGA CTG CTC; XBP-1 5⬘-GCT CAG ACT GCC AGA
GAT CG; XBP-1 3⬘-GTC CSG AAT GCC CAA CAG G; Bcl-6 5⬘-CTG
ACA GCT GTA TCC AGT TCA CC; Bcl-6 3⬘-TCT TGG GGC ATC
AGC ATC.
Expression of Blimp-1 and Pax-5 protein by activated human memory
B cells was also determined. B cell subsets were isolated by cell sorting,
and then lysed in ice cold lysis buffer (10 mM Tris-HCl (pH 7.8), 1%
Nonidet P-40, 150 mM NaCl, and enzyme inhibitors). Cell lysates were
electrophoresed through 12% acrylamide gels containing 0.1% SDS and
transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed with Abs against Blimp-1 (31), Pax-5 (C-20; Santa
Cruz Biotechnology) or SHP-2 (Santa Cruz Biotechnology) followed by
HRP-conjugated anti-rat, anti-goat, and anti-rabbit Ig antiserum, respectively (all from Santa Cruz Biotechnology). The membranes were developed using ECL (Pierce) and autoradiography.
Chemotaxis assays
Migration assays of activated memory B cells were performed using 5-␮m
Costar Transwell plates (Corning). CXCL12 (100 ng/ml), CXCL13 (1000
ng/ml), or CCL21 (600 ng/ml) (32) were diluted in RPMI 1640 containing
0.5% BSA (chemotaxis medium) and added to wells of a 24-well plate in
600 ␮l. Chemotaxis medium was used as a control for basal cell migration.
CFSE-labeled memory B cells were activated with CD40L, IL-2, and IL-10
for two lots of 4 days, then were washed, and were resuspended in chemotaxis medium, and 5 ⫻ 105 cells were added to the upper chamber of the
Transwell in 100 ␮l. Plates were incubated for 4 h at 37°C. A known
number of Calibrite beads was then added to each bottom well before
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FITC-conjugated anti-CD20 and PE-conjugated anti-CD19 and CD45
mAb were purchased from BD Biosciences; anti-CCR7, PE-conjugated
isotype controls, CD21, CD27, CD80, CD86, CD95, and CXCR4 mAb,
biotinylated anti-CD44, anti-hamster IgG, and streptavidin (SA)-conjugated to PerCp were purchased from BD Pharmingen. PE-anti-CD22,
CD23, CD31, CD38, CD62L, CD138, HLA-DR mAb, biotinylated IgG1
isotype control, and anti-CD38 mAb, allophycocyanin-conjugated isotype
control, anti-CD20 and anti-CD38 mAb, and SA-allophycocyanin were
purchased from Caltag Laboratories; biotinylated anti-CXCR5 mAb was
from R&D Systems; biotinylated anti-human CD27 mAb was from eBioscience; PE-conjugated anti-CD40 mAb (mAb89) was provided by J.
Banchereau (Schering Plough Labs, Dardilly, France); PE-conjugated antiCD39 (A1) mAb has been described previously (26). Purified and biotinylated F(ab⬘)2 of goat anti-human IgM, IgG, or IgA polyclonal Ab were
purchased from Southern Biotechnology. Recombinant human BAFF, and
mouse anti-BAFF receptor (anti-BAFF-R; clone 9.1), anti-transmembrane
activator of and CAML interactor (TACI; clone C4D7) and hamster anti-B
cell maturation Ag (BCMA; clone C4E2.2) mAbs were provided by Dr. S.
Kalled (Biogen Idec, Cambridge, MA; Ref. 25). Recombinant CD40L expressed as membranes in Sf21 insect cells infected with baculovirus vector
containing human CD40L cDNA was provided by Dr. M. Kehry (Boehringer
Ingleheim, Ridgefield, CT). IL-2 was purchased from Endogen; IL-10 was
provided by Dr. R. de Waal Malefyt (DNAX Research Institute, Palo Alto,
CA). CFSE was from Molecular Probes. Recombinant human CXCL12 and
CCL21 were purchased from PeproTech; CXCL13 was from R&D Systems.
4036
INCREASED EXPRESSION OF CD27 SHOWS PC LINEAGE-COMMITTED CELLS
harvesting, and the number of B cells that had migrated was calculated.
The migrated population was stained with anti-CD27 and CD38 mAb to
determine the proportions of different populations of activated memory B
cells which were then used to calculate the absolute number of cells of each
population that had migrated. Migration was calculated as the percentage
of input cells.
Results
CD27 is up-regulated on activated human memory B cells in
vitro in an IL-10-dependent and division-linked manner
FIGURE 1. Up-regulation of CD27 on activated memory B cells is
IL-10 dependent and increases with cell division. CFSE-labeled human
splenic memory B cells were cultured with CD40L alone or with IL-10, or
IL-2 and IL-10 for 5 days. After this time, the expression of CD27 (a) and
CD38 (b) was determined in the context of cell division (i.e., vs CFSE).
The values in all panels represent the mean percentage of cells (⫾SEM)
expressing CD38 or CD27 from four independent experiments. c, Coexpression of CD38 and CD27 by memory B cells activated with CD40L,
IL-2, and IL-10 was determined according to division history by gating on
consecutive divisions. The percentages of cells with distinct phenotypes in
divisions 3–7 is indicated.
Proliferating B cells expressing increased levels of CD27 are
enriched for ISC
The ability of distinct populations of activated B cells, defined by
division history and differential expression of CD27 and CD38, to
produce Ig was next determined. For these studies, B cells were
initially resolved into populations that had undergone the following: ⬍3 divisions (population 1); several rounds of division and
remained CD38⫺ (population 2); or undergone the same number
of cell divisions as population 2 yet had differentiated to become
CD38⫹ (population 3; see Fig. 1b; Refs. 17 and 25). ISC increased
in frequency in populations 2 (CD38⫺) and 3 (CD38⫹) of activated memory B cells, compared with population 1 (Fig. 2a) (17).
However, these populations were heterogeneous as demonstrated
by ⬍40% of them secreting IgM, IgG, and IgA (Fig. 2a). Further
heterogeneity was apparent from the finding that within the
CD38⫺ and CD38⫹ populations in the later divisions, there were
cells that expressed different levels of CD27 (i.e., low or high; Fig.
1c). To determine whether the ISC in populations 2 and 3 were
partitioned into subsets expressing different levels of CD27, population 1, population 2 CD27low, population 2 CD27high, population 3 CD27low, or population 3 CD27high were examined by
ELISPOT. For both populations 2 and population 3, ISC were
enriched in the CD27high subset, containing up to 5-fold more ISC
than the CD27low subset (Fig. 2b). This analysis revealed that up
to 80% of these cells were ISC, an ⬃2-fold increase over the total
population 2 or 3 (compare Fig. 2, a and b).
To extend these findings, the five different populations of activated human memory B cells were sort-purified and recultured for
a further 2 days with CD40L, IL-2, and IL-10, and the amount of
secreted Ig was determined. Consistent with the ELISPOT data,
population 1 B cells secreted very little Ig, while population 3
CD27high B cells secreted the greatest amounts of Ig, exceeding
that by the other populations by 2- to 10-fold (Fig. 2c). Population
2 CD27high B cells produced significant amounts of Ig, albeit less
than those produced by population 3 CD27high B cells (Fig. 2c).
Population 2 B cells undergo less proliferation and greater apoptosis than population 3 B cells (17, 20, 24, 25)—this is the likely
explanation for the reduced levels of Ig detected in cultures of
these cells following the secondary culture compared with population 3 CD27high B cells, and the apparent disparity between the
data obtained for ELISPOT and ELISA analysis of these cells (Fig.
2c). Thus, up-regulation of CD27 on divided memory B cell blasts
facilitates the reliable detection of the predominant populations of
ISC present in these cultures, while coexpression of CD38 and
increased CD27 identifies cells capable of secreting the greatest
amounts of Ig.
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Human PC express a higher level of CD27 than memory B cells (3,
6, 9, 14, 33). Therefore, it was of interest to investigate the expression of CD27 on memory B cells cultured under conditions
that induce their proliferation and differentiation to CD38⫹ B cells,
which contain a population of ISC. Expression of CD27 on
CD40L-stimulated memory B cells did not greatly change with
division (Fig. 1a, left panel), remaining at a level comparable to
cells before culture (data not shown). Addition of IL-10 alone or in
combination with IL-2 resulted in the appearance of a population
of cells expressing an increased level of CD27 (Fig. 1a, middle and
right panels). Notably, this population increased in frequency with
cell division in a manner analogous to the appearance of the
CD38⫹ cells (Fig. 1, a and b).
It was next determined whether CD38⫹ B cells generated from
memory B cells coexpressed elevated levels of CD27, or whether
a CD27highCD38⫺ population was also generated. Because the increase in expression of both CD27 and CD38 was division-linked
(Fig. 1, a and b), this analysis was performed by assessing their
expression on cells in different divisions. Cells that had undergone
⬍2 divisions (i.e., division 0 –2) expressed uniform levels of CD27
and CD38. However, after approximately the third division, a population of cells appeared that had up-regulated expression of these
molecules (Fig. 1c). With each subsequent division, multiple subsets of cells could be resolved such that in later divisions cells were
either CD38⫹CD27high, CD38⫾CD27high, CD38⫹CD27low, or
CD38⫾CD27low (Fig. 1c). Thus, when memory B cells are stimulated with T cell help, they are capable of generating numerous
populations of cells discernible by division history and the differential expression of CD27 and CD38.
The Journal of Immunology
4037
FIGURE 2. ISC are enriched in populations expressing increased levels of CD27. a and b, CFSE-labeled memory B cells were cultured with CD40L,
IL-2, and IL-10 for 4 days, washed, and then re-cultured for an additional 4 days under the same conditions. Activated B cells defined as population 1, 2,
or 3 (a, see Fig. 1a), or population 1 (b), population 2 CD27low or CD27high, or population 3 CD27low or CD27high were sorted into ELISPOT plates
precoated with Ig H chain specific Ab and the proportion of cells secreting IgM (䡺), IgG (f), or IgA (u) determined. The results presented in a and b
are from the same experiment. c, CFSE-labeled memory B cells were cultured with CD40L, IL-2, and IL-10 for 5 days. Cells defined according to division
history and expression of CD27 and CD38 were sort-purified and recultured for an additional 2 days with CD40L, IL-2, and IL-10. The amount of Ig
secreted was determined by Ig H chain specific immunoassays. The values represent the mean Ig production from each population of activated memory
B cells from three independent experiments.
In addition to increasing expression of CD38 and CD27, human
PC alter the expression of other molecules such that their phenotype is distinct from that of naive or memory B cells. Compared
with mature B cells, primary PC down-regulate CD20, CD21,
CD22, CD84, BAFF-R, CXCR5, CCR7, and, to a lesser extent,
CD19, CD40, and HLA-DR (3, 32). In contrast, expression of CD31,
CD39, CD44, CD49d, CD86, and CD95 is increased while CD45
and CXCR4 remain unchanged (3, 32). Similarly, BM PC acquire
expression of CD138 (5). To extend the characterization of ISC
generated from memory B cells in vitro, we determined the phenotype of activated cells corresponding to population 1, and pop-
ulations 2 and 3 that were either CD27low or CD27high. The phenotype of the ISC (i.e., CD27high populations 2 and 3) bore a
striking resemblance to each other as well as to in vivo-derived PC
inasmuch that expression of CD19, CD20, CD21, CD22, CD40,
CD84, CXCR5, HLA-DR, and BAFF-R were reduced compared
with the nonsecreting cell in population 1 as well as the CD27low
subsets of populations 2 and 3 (Fig. 3; Table I). Similarly, CD39,
CD95 (Table I), CD86, CD44, CD49d, and CD62L (Fig. 3) were
uniformly expressed on CD27high ISC in populations 2 and 3 while
expression of these molecules on the CD27low B cells was heterogeneous, with bimodal expression of some molecules being detected. CD31 and BCMA were weakly induced on activated B
FIGURE 3. Memory B cells alter their phenotype during in vitro differentiation. CFSE-labeled human memory B cells were cultured as described for
Fig. 2a. The cells were incubated with anti-CD27 and CD38 mAbs, and the expression of the indicated molecule on cells corresponding to population 1,
population 2 CD27low or CD27high, and population 3 CD27low or CD27high was assessed by flow cytometry. For each plot, the thick and thin histograms
represent the fluorescence of cells incubated with the specific or control mAb, respectively. Fluorescence was measured on a log scale.
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Phenotypic characterization of plasmablasts generated in vitro
4038
INCREASED EXPRESSION OF CD27 SHOWS PC LINEAGE-COMMITTED CELLS
Table I Memory B cells alter their phenotype during in vitro proliferation and differentiation to IGSa
Activated Memory B cell Population (mean fluorescence intensity)
No. 1
Isotype Ctl
CD19
CD20
CD21
CD22
CD31
CD39
CD40
CD45
CD80
CD84
CD86
CD95
CD138
HLA-DR
Isotype Ctl
CXCR5
5.2
584
363
160.1
683
6.71
568
459
268
150
34.8
81.2
203.5
6.0
492
16.0
33.5
No. 2 CD27
low
4.1
330
134
31.5
237
6.1
334
220
150
91
13
114.5
116.9
5.0
320
5.7
5.95
No. 2 CD27high
No. 3 CD27low
No. 3 CD27high
4.5
237
52
18.8
213
7.8
685
164
178
90
8.6
179.2
147.3
5.4
197
7.07
6.8
5.0
97
36
13.5
57.7
10.3
336
84.1
123
40.5
6.5
105.4
97.3
6.1
263
7.13
7.5
5.4
140
19
13.4
57.2
10.5
743
88.6
144
45.5
6.5
164.8
142.4
6.4
128
8.3
8.0
cells in populations 2 and 3, while expression of CD45 and
CXCR4 was similar on all populations (Fig. 3, Table I), consistent
with the phenotype of primary PC (3, 5, 32). No induction of
CD138 expression was detected on the activated splenic memory
B cells (Table I), analogous to its absence from primary PC in
human spleen (3) and some in peripheral blood and tonsil (5).
Collectively, the phenotypes of CD27high cells in populations 2
and 3 are very similar, consistent with the functional similarities of
these cells (Fig. 2b). Remarkably, the phenotype of population 1 B
cells was similar to resting human memory B cells (Fig. 3, Table
I; Refs. 3 and 26), suggesting that these cells remained nondifferentiated with the potential to expand the memory B cell pool.
Differential expression of transcription factors by distinct
populations of activated B cells
Differentiation of mature B cells into ISC is accompanied by
changes in expression of specific transcription factors. For instance, while Pax-5 is expressed by naive and memory B cells, its
expression is extinguished in PC. Similarly, expression of Bcl-6 is
restricted to germinal center B cells (34) while Blimp-1 and XBP-1
are induced at the PC stage (3, 23, 35–37). Therefore, expression
of these transcription factors during the in vitro differentiation process that yields ISC from memory B cells was assessed. Analysis
of activated memory B cells revealed that expression of Pax-5 was
maintained in population 1 (Fig. 4a, lane 2), but decreased in cells
that had undergone further proliferation (Fig. 4a, lane 3– 6). Interestingly, while Pax-5 was absent from population 3 CD27high B
cells, it remained detectable in the CD27low subset of population 3
(Fig. 4a, lane 5). It is possible that the heightened expression of
Pax-5 by these cells prevents them from becoming efficient ISC
(Fig. 2), because Pax-5 may counter the inductive effect of XBP-1
on the differentiation of these cells toward an ISC fate (23). In
contrast to Pax-5, Blimp-1 (Fig. 4b) and XBP-1 (Fig. 4c) were low
in population 1 (lane 2) and incrementally increased as the B cells
underwent proliferation and differentiation to an effector ISC
(lanes 3– 6), such that CD27high cells within population 3 expressed the highest levels of these genes. Bcl-6 was induced in
activated B cells, down-regulated in population 2 and again increased as the cells acquired expression of CD38 to become population 3 (Fig. 4d).
The differential expression of Pax-5 and Blimp-1 by the functionally distinct subsets of activated B cells was also examined at
the protein level. For these experiments, activated memory B cells
were sorted into: population 1, population 2 CD27low and
CD27high, as well as population 3 CD27high. Insufficient numbers
of population 3 CD27low cells were collected, thereby precluding
analysis of these cells. Pax-5 and Blimp-1 were expressed in unsorted activated memory B cells (Fig. 4, f and g, lane 1), consistent
with previous reports (38, 39). However, analysis of these proteins
in the populations of sorted B cells demonstrated that Pax-5 was
dramatically reduced once the memory B cells became CD27high
(Fig. 4f, lanes 2–5). In contrast, expression of Blimp-1 was low to
absent in population 1 B cells (Fig. 4g, lane 2), but increased once
FIGURE 4. In vitro-generated ISC express transcription factors consistent with commitment to the PC lineage. Population 1, population 2
CD27low or population 2 CD27high, population 3 CD27low, or population 3
CD27high B cells were isolated by sorting. a–e, RNA was extracted from
the sort-purified populations and transcribed into cDNA. The amounts of
cDNA were then normalized for expression of GAPDH (bottom panel; e)
and used as template to determine the relative expression levels of Pax-5
(a), Blimp-1 (b), XBP-1 (c), and Bcl-6 (d) by semiquantitative PCR. Molecular grade dH2O (lane 1) was used as a negative control. f– h, Unsorted
(total B cells) or sort-purified populations of activated B cells were solubilized in lysis buffer, and expression of Pax-5 (f) and Blimp-1 (g) was
determined by Western blotting using specific Ab. h, Membranes were also
probed with anti-SHP-2 Ab to demonstrate similar protein loading.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a
CFSE-labeled human memory B cells were cultured with CD40L, IL-2, and IL-10 for 4 days, washed, and then recultured under the same conditions for a further 4 days.
After this time, the cells were harvested and incubated with anti-CD27 and CD38 mAbs, and the expression of the indicated molecule on B cells corresponding to population
1, population 2 CD27low or CD27high, and population 3 CD27low or CD27high was assessed by flow cytometry. The values represent the mean fluorescence intensity of expression
of the indicated molecule for all B cells. These results are representative of at least three independent experiments.
The Journal of Immunology
4039
the cells underwent substantial cell division (Fig. 4g, lane 3–5).
These results confirm the differences observed at the mRNA level
(Fig. 4, a and f; b and g). Thus, the differential expression of
transcription factors by phenotypically distinct populations of activated human memory B cells correlated with differences in function, with the ISC populations expressing increased levels of
Blimp-1 and XBP-1, and lower levels of Pax-5.
Effect of BAFF and CD70 on the generation of different
populations of activated memory B cells
FIGURE 6. In vitro-generated ISC retain responsiveness to CXCL12,
but not CXCL13 or CCL21. CFSE-labeled memory B cells were cultured
as described for Fig. 2a. Activated memory B cells (5 ⫻ 105/well) were
loaded into the upper chamber of a Transwell, and medium alone, CXCL13
(1000 ng/ml), CXCL12 (100 ng/ml), or CCL21 (600 ng/ml) were added to
the lower wells. Cells were allowed to migrate for 4 h at 37°C. Migrated
cells were harvested from the lower wells and stained with anti-CD27 and
CD38 mAb, enabling resolution of population 1, population 2 CD27low or
CD27high, or population 3 CD27low or CD27high B cells. The different symbols (E, F) represent the percentage of cells migrating in two different
experiments; the columns represents the mean of the individual values.
Altered responsiveness to lymphoid chemokines characterizes
the generation of ISC
A characteristic of B cell development and differentiation is the
alteration in responsiveness to chemokines. For instance, B cells
increase responsiveness to the lymphoid chemokines CXCL12,
CXCL13, and CCR7 ligands (CCL19, 21) as they develop from
pro-B cells in the BM through to follicular B cells in the spleen
(40). In contrast, PC lose responsiveness to CXCL13 and CCR7
ligands, yet retain the ability to respond to CXCL12 (32, 41, 42).
Given the alteration in expression of receptors for these chemokines on the different populations of activated memory B cells, the
chemotactic responses of these populations was next examined.
Population 1 B cells exhibited chemotactic responses to CXCL12,
CXCL13, and CCL21 (Fig. 6). As the activated memory B cells
underwent proliferation and differentiation to ISC, their responsiveness to CXCL13 and CCL21 gradually declined such that population 3 CD27high B cells were almost unresponsive to these chemotactic ligands (Fig. 6). In contrast, activated B cells continued to
respond to CXCL12, with an increased response being evident for
population 3 CD27high B cells (Fig. 6). Thus, in vitro-generated
ISC behave analogously to primary PC with respect to chemotactic
responses.
Discussion
FIGURE 5. BAFF and CD70 increase the recovery of Ig-secreting effector cells generated from memory B cells. CFSE-labeled memory B cells
were initially cultured with CD40L, IL-2, and IL-10 for 4 days, harvested,
washed, and then recultured with IL-2 and IL-10 in the absence (䡺) or
presence of BAFF (f) (a), fixed untransfected P815 cells (u) (b), or fixed
CD70-expressing P815 cells (CD70 Tf; f). After a further 4 days, the total
number of cells corresponding to population 1, population 2 CD27low and
CD27high, and population 3 CD27low and CD27high was calculated by multiplying total cell number by the frequency of each of these cell subsets. The
values in a represent the mean ⫾ SEM of three independent experiments;
those in b are representative data from one of two separate experiments.
The generation of ISC from precursor cells is a complex and
highly regulated process. Here, we have tracked in detail the in
vitro differentiation of human memory B cells into effector cells
capable of secreting Ig. Different stages of differentiation could be
resolved by concomitantly comparing the division history of activated B cells and surface expression of CD27 and CD38 (Figs. 1,
2, and 7). Although CD27 is constitutively expressed by human
memory B cells (26, 28), it was up-regulated on some of them as
they proliferated in response to CD40L and IL-10, but not CD40L
alone (Fig. 1). This parallels our previous observations of divisionlinked acquisition of CD38 expression by memory B cells stimulated with CD40L and IL-10, and CD138 on stimulated murine B
cells, and the subsequent generation of ISC (17, 25, 43). Our data
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The number of B cells in populations 2 and 3 can be influenced by
CD40L or BAFF in the secondary cultures. CD40L is critical for
the persistence of population 2 B cells, and can increase population
3 B cells, while BAFF preferentially favors the survival of population 3 (17, 25). Interestingly, culture of activated memory B cells
with CD27 ligand (CD70), another TNF superfamily molecule,
can increase the proportion of CD38⫹ B cells generated from
memory B cells (30). However, the effect of CD70 on the number
of surviving B cells, or different subsets of activated B cells, has
not been examined. Therefore, we investigated whether the different B cell populations preferentially responded to secondary stimulation through different BAFF receptors or CD27.
BAFF increased the survival of population 2 CD27high B cells,
as well as both subsets of population 3, yet had no significant effect
on population 1 nor the CD27low subset of population 2 (Fig. 5a).
However, the greatest effect of BAFF was on population 3
CD27high B cells, where almost 5-fold more cells were generated
compared with secondary cultures containing IL-2 and IL-10 alone
(Fig. 5a). Consequently, in the presence of BAFF, this population
of cells dominated the culture, comprising ⬎50% of total cells,
compared with cultures containing IL-2 and IL-10 alone, where the
CD38⫹CD27high cells represented ⬍30%. When activated memory B cells were cultured with CD70 transfectants, there was an
increase in the number of population 1 B cells, as well as the
CD27high cells in populations 2 and 3 (Fig. 5b). Consistent with the
down-regulation in CD27 expression, there was a lesser effect on
the CD27low B cells (Fig. 5b). The ability of CD70-expressing
transfectants to increase the number of CD27high B cells (i.e., ISC)
correlated with increased Ig production in these cultures (data not
shown). Thus, both BAFF and CD70 are capable of increasing the
generation of ISC from memory B cells.
4040
INCREASED EXPRESSION OF CD27 SHOWS PC LINEAGE-COMMITTED CELLS
BAFF (3, 24, 25, 32), revealing them to be terminally differentiated. In contrast, in vitro-derived CD38⫹CD27high ISC likely correspond to plasmablasts, such as those detected in reactive plasmacytosis, or following infection (21) (Fig. 7). The similarities
between plasmablasts generated in vitro from activated human
memory B cells and those detected in vivo with respect to cell
surface phenotype, expression of transcription factors, proliferation and function are remarkable (compare Fig. 3, Table I to Ref.
21), and thus illustrates the validity of our in vitro culture system
to examine the processes involved in this aspect of human B cell
differentiation.
Another novel finding in our study was the identification of a
CD38⫺CD27high population with a phenotype and capacity to secrete Ig that was identical with the CD38⫹CD27high B cells (Figs.
2 and 3). Despite these similarities, some important differences
were found between these two populations of in vitro-derived ISC.
First was their responsiveness to BAFF. This current study refined
our previous analysis (25) by revealing that the greatest effect of
BAFF was on the viability of CD38⫹CD27high ISC, while its effects on CD38⫺CD27high ISC were modest (Figs. 5 and 7). Second, CD38⫹ B cells proliferate at a greater rate than CD38⫺ B
cells (17, 20, 25), revealing CD38⫹CD27high ISC to be plasmablasts. A molecular explanation for this difference may be attributable to differential expression of Bcl-6. CD38⫺ B cells (Population 2) expressed less Bcl-6 than CD38⫹ B cells (population 3;
Fig. 4). Interestingly, Bcl-6 can regulate the cell cycle by controlling expression of genes such as c-myc (46). This is further exemplified by the finding that the frequency of proliferating cells in
Bcl-6 transgenic mice is markedly enhanced compared with control mice (47), and that overexpression of dominant-negative Bcl-6
arrested proliferation of a human B cell line (46). Thus, increased
expression of Bcl-6 by CD38⫹ B cells may provide them with the
molecular machinery required for differentiation into rapidly dividing plasmablasts. An additional explanation for the differences
between the CD38⫺CD27high and CD38⫹CD27high ISC may be
that these cells arise from different progenitor memory B cells,
namely IgM-expressing or Ig isotype switched memory cells (24,
FIGURE 7. Model of terminal B cell differentiation. In human lymphoid tissues, naive B cells become activated by Ag and costimulatory factors and
form a germinal center where mutation of Ig V region genes and Ig isotype switching occurs. Upon exiting the germinal center, B cells differentiate into
either memory B cells or early plasmablasts, identified by increased expression of CD27. Plasmablasts can also rise directly from memory B cells upon
re-exposure to specific Ag. Up-regulation of CD38 characterizes further differentiated (“late”) plasmablasts. These cells can migrate to the BM, gut, red
pulp of spleen, or mucosal epithelium of tonsil under the direction of specific chemokines (CXCL12, CCL25, CCL28; see Ref. 59). Once in these
microenvironmental niches, ISC compete for survival signals facilitating differentiation into long-lived PC (see Refs. 23, 32, and 59). Factors identified that
support the survival of (i.e., CD40L, CD70, BAFF, IL-6, stromal cells), and the phenotypes and transcription factors expressed by, each stage of terminal
B cell differentiation are indicated. Some memory B cells can also yield alternative phenotypic and functional states which may correspond to memory B
cell precursors that replenish the memory B cell population (detailed in Discussion).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
also demonstrated that the frequency of CD38⫹CD27high B cells
appearing in cultures of stimulated memory B cells increased with
cell division (Fig. 1c). Furthermore, these cells secreted high Ig
levels (Fig. 2) and expressed a transcriptional profile comparable
to primary PC (Fig. 4). Thus, this phenotype reliably identifies ISC
generated in vitro and allows comparison between human ISC generated in vivo and in vitro (Fig. 7).
In addition to increased production of Ig, the CD38⫹CD27high B
cells exhibited several other features of primary PC. First, similar
to PC in spleen, tonsil, and PB, CD38⫹CD27high ISC generated in
vitro exhibit reduced expression of CD20, CD21, CD22, CXCR5,
and BAFF-R; sustained expression of CD19, CD39, CD40, CD45,
CD86, CXCR4, and HLA-DR; and lacked expression of CD138
(Fig. 3, Table I; Refs. 3, 5, 32, and 44). Second, CD38⫹CD27high
ISC retained responsiveness to CXCL12, while responses to
CXCL13 and CCL21 were greatly diminished (Fig. 6), akin to
primary PC (32, 41, 42). Lastly, expression of Blimp-1 and XBP-1
was greatest, and Pax-5 extinguished, in in vitro CD38⫹CD27high
ISC compared with other populations of activated B cells (Figs. 4
and 7). Similar to alterations in surface phenotype, these molecular
changes recapitulate the differential expression of Pax-5, Blimp-1
and XBP-1 observed for mature B cells and primary PC (3, 5, 7,
36, 38), and complement our recent report of gene expression by
ISC generated from mouse B cells in vitro in response to T celldependent stimuli (43). Similar observations regarding expression
of transcription factors have been obtained for human B cells activated in vitro to differentiate toward ISC (38, 39), or in murine
splenic B cells following immunization (45). However, a disadvantage of these previous studies was that they examined bulk
populations of activated B cells, rather than distinct subsets, and
did not correlate mRNA levels with protein (38, 39, 45). Consequently, it was unclear in which cells these transcriptional changes
were occurring. Notably, the proliferative activity (17, 20), coupled with Bcl-6 expression and responsiveness to BAFF (Figs. 4
and 5; Ref. 25) also distinguished in vitro CD38⫹CD27high ISC
from primary human PC. The latter population contains noncycling cells which lack expression of Bcl-6, and do not respond to
The Journal of Immunology
type. We propose that human ISC can be resolved by increased
expression of CD27, while the coexpression of CD38 delineates
the ISC population into an early stage of commitment to the ISC
lineage (CD38⫺) and plasmablasts (CD38⫹) (Fig. 7). These findings will further our understanding of the complex regulation of
the pathways B cells undergo during differentiation to an
effector cell.
Acknowledgments
We thank Drs. Marylin Kehry, Rene de Waal Malefyt, and Susan Kalled
for generously providing reagents, the Australian Red Cross Blood Service
for providing human spleens, Tara Macdonald and Joseph Webster for cell
sorting, and Prof. Tony Basten for critical review of this manuscript.
Disclosures
The authors have no financial conflict of interest.
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4041
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INCREASED EXPRESSION OF CD27 SHOWS PC LINEAGE-COMMITTED CELLS
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In Results, Figure 1, panels a and b were reversed. The error has been corrected in the online version, which now differs
from the print version as originally published. The correct figure is shown below.
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