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Circulating human B and plasma cells. Age-associated changes incounts and detailed characterization of circulating normal CD138and CD138+ plasma cells
by Anouk Caraux, Bernard Klein, Bruno Paiva, Caroline Bret, Alexander Schmitz,Gwenny M. Fuhler, Nico A. Bos, Hans E Johnsen, Alberto Orfao,and Martin Perez-Andres
Haematologica 2010 [Epub ahead of print]
doi:10.3324/haematol.2009.018689
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Copyright 2010 Ferrata Storti Foundation.Published Ahead of Print on January 15, 2010, as doi:10.3324/haematol.2009.018689.
DOI: 10.3324/haematol.2009.018689
Circulating human B and plasma cells. Age-associated changes in counts
and detailed characterization of circulating normal CD138- and CD138
+
plasma cells
Running title: Blood B-lymphocytes and plasma cells in adults
Anouk Caraux,1 Bernard Klein,1-3 Bruno Paiva,4,5 Caroline Bret,1-3 Alexander Schmitz,6
Gwenny M. Fuhler,7 Nico A. Bos,
7 Hans E Johnsen,
6 Alberto Orfao,5,8 and Martin Perez-
Andres5,8 for the Myeloma Stem Cell Network (MSCNET)
1INSERM, U847, Montpellier, F-34295 France; 2CHU Montpellier, Institute of Research
in Biotherapy, F-34295 France; 3Université Montpellier1, F-34967 France; 4Service of
Hematology, Hospital Universitario de Salamanca, Salamanca, Spain; 5Centro de
Investigación del Cáncer, University of Salamanca-CSIC, Salamanca, Spain; 6Service of
Hematology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark;7University Medical Center, Groningen, Netherlands; 8Service of Cytometry, Department of
Medicine, University of Salamanca, Salamanca, Spain
FundingThis work was supported by grants from the Ligue Nationale Contre le Cancer (équipelabellisée 2009), Paris, France, from INCA (n°R07001FN), the Fondo de InvestigaciónSanitaria, Ministerio de Ciencia e Innovación (FIS 06-0824), Madrid, Spain, GerenciaRegional de Salud de Castilla y León (GRS206/A/08),Valladolid, Spain, the AYUDAPARA LA FINANCIACIÓN DE LOS PROGRAMAS DE ACTIVIDADINVESTIGADORA DE LOS GRUPOS DE INVESTIGACIÓN DE EXCELENCIA DECASTILLA Y LEÓN (EDU/894/2009, GR37), Junta de Castilla y León, Valladolid, theInstituto de Salud Carlos III, Ministerio de Ciencia e Innovación (RTICCRD06/0020/0035), Madrid, Spain, and from MSCNET European strep (N°E06005FF)Cancer Centers research network.
Correspondence: Alberto ORFAO and Bernard KLEIN
Prof. Bernard Klein
INSERM U847, Institute for Research In Biotherapy, CHU Montpellier, Hospital St Eloi,
Av Augustin Fliche, 34295 Montpellier. FRANCE. Phone number: +33 467 330 455, Fax
number: +33 467 330 459. E-mail:bernard.klein@inserm.fr
Prof. Alberto Orfao
Centro de Investigación del Cáncer Avda. Universidad de Coimbra S/N, Campus Miguel de
Unamuno, 37007-Salamanca. SPAIN. Phone number: +34 923 294 811, Fax number: +34
923 294 795. E-mail:orfao@usal.es
Abbreviations used in this paper
PC, plasma cells; HDs, healthy donors; BM, bone marrow; PB, peripheral blood; DC,
dendritic cells; HSC, hematopoietic stem cells; sIg, surface Ig; Ig!, Ig light chain !; Ig", Ig
light chain "; MFI, mean fluorescence intensity; cyIg, cytoplasmic Ig; SI, staining index.
DOI: 10.3324/haematol.2009.018689
2
ABSTRACT
Generation of B and plasma cells (PC) involves several organs with a necessary cell
trafficking between them. A detailed phenotypic characterization of four circulating B-cell
subsets - immature-, naïve-, memory- B-lymphocytes and PC – of 106 healthy adults was
realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the
minimal combination of subsetting markers allowing unequivocal identification of
immature (CD10+CD27
-CD38
+, 6±6 cells/!l), naïve (CD10
-CD27
-CD38
-, 125±90 cells/!l),
memory B-lymphocytes (CD10-CD27
+CD38
-, 58±42 cells/!l), and PC (CD10
-
CD27++
CD38++
, 2.1±2.1 cells/!l) within circulating CD19+ cells. From these four subsets,
only memory B-lymphocytes and PC decreased with age, both in relative and absolute
counts. Circulating PC split into CD138- (57%±12%) and CD138
+ (43%±12%) cells, the
latter displaying a more mature phenotypic profile: absence of surface immunoglobulin,
lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and
CD27. Unlike B-lymphocytes, both populations of PC are KI-67+ and show weak CXCR4
expression.
DOI: 10.3324/haematol.2009.018689
3
INTRODUCTION
Human B cell biology has been extensively documented.(1)
Circulating human B
cells comprise two thirds of CD27-CD20
+CD19
+CD38
- naïve B-lymphocytes and one third
of CD27+CD20
+CD19
+CD38
- memory B cells. Very low numbers of plasma cells (PC,
2/µL) are found in peripheral blood (PB) of healthy donors (HDs). Because of their low
count, only few studies were devoted characterizing their phenotype, most of them dealing
with newly generated PC after in vivo immunization.(2)
Steady-state circulating PC lack
CD20, express CD19 and CD38high
. It has been recently reported that steady-state
circulating PC are mainly of mucosal origin, the majority of them secreting IgA (84%),
expressing CCR10 (56%) and #7 integrin (32%).(3)
Steady-state circulating PC are
generally termed plasmablasts because only half express CD138, a proteoglycan that is a
hallmark of PC(4)
, while they are CD45+ and HLA-class II
+. Plasmablasts are generated in
the lymph nodes, and induced to circulate for a short period until they will reach a niche in
BM, spleen, mucosa associated lymphoid tissues (MALT) or lymph nodes.(5)
These niches
will provide to circulating early PCs those factors required to survive and to further
differentiate into long-living mature PC.(1)
In murine BM, PC niche involves SDF-1
producing cells and is shared with hematopoietic stem cells (HSC) and pro-pre B-cells.(1)
The rarity of this niche is a matter of regulation of normal Ig production.(6)
In particular,
new-born plasmablasts, generated after in vivo Ag immunization, have to compete with old
PC for binding to a niche, inducing the old PC to recirculate.(7)
Another minor population of circulating B-cells which accounts for 2-4% of all
PB B-cells has been documented(8)
: transitional or immature B-cells. These cells have an
immature phenotype (CD10+, CD24
high, CD38
high), unmutated Ig genes and a reduced
DOI: 10.3324/haematol.2009.018689
4
ability to be activated in vitro.(8, 9)
Notably, these immature B-cells appear first in PB after
HSC allograft(8)
and their frequency is highly increased in cord blood.(8, 9)
Recently,
additional heterogeneity has been reported for human transitional B cells with a more
differentiated stage expressing ABCB1 transporter and intermediate density of CD10 and
CD38.(10, 11)
There is a progressive defect to mount high affinity humoral immune responses in
elderly people.(12)
This defect implies several mechanisms: i) a decrease in BM niches able
to support B-cell generation and PC survival, due to the progressive replacement of
hematopoietic BM by fat cells; ii) a defect in germinal centers due to a decreased follicular
DC function and T-cell senescence; iii) a defect of B-cells to undergo Ig class switch
recombination and somatic mutation due to reduced E47 and AID gene expressions.(13)
In this study we have first characterized the above listed populations of circulating
B-cells using multi-parameter flow cytometry, with the aim to define the best combination
of markers to identify them, and to study their fluctuation with age. In addition, we have
characterized in detail the steady-state circulating PC as regards to their activation status
and homing phenotype.
DOI: 10.3324/haematol.2009.018689
5
DESIGN AND METHODS
Detailed methodologies are described in the Supplementary Appendix.
RESULTS AND DISCUSSION
Immunophenotypic characteristics of human peripheral blood B-cell subsets
Circulating B-cells in a given individual are important indicators of the state of B-
cell production because generation of B-lymphocytes and PC involves sequential
maturation steps in different organs and tissues(14)
and a necessary cell traffic between these
organs through PB.(15)
Varying strategies have been applied for their identification and no
study has comparatively analyzed these four B-cell subsets in large cohorts of healthy
donors (HDs). Here, we show that four B-cell subsets were systematically identified in PB
of 106 HDs showing phenotypic profiles of immature (CD10+CD19
+CD20
+CD27
-
CD38+)
(8), naïve (CD10
-CD19
+CD20
+CD27
-CD38
-)
(1), and memory (CD10
-
CD19+CD20
+CD27
+CD38
-) B-lymphocytes
(1), in addition to PC (CD10
-CD19
+CD20
-
CD27++
CD38++
)(1)
. Principal component analysis showed that CD10, CD27, and CD38 are
the minimal marker combination for unequivocal identification of immature, naïve, and
memory B-lymphocytes, as well as PC among CD19+ B-cells (Figure 1A). Compared to
naïve B-lymphocytes, circulating immature B-lymphocytes, previously described as
phenotypically similar to transitional murine B-cells(8)
, retain a phenotype of late BM B-
cell precursors (i.e. CD5, CD10, and CD38++
) (Supplementary Figure S1), supporting they
are immature B-lymphocytes leaving the BM prior to full maturation.(8)
Transition from
naïve to memory B-lymphocytes was characterized by increased CD24, CD25, CD27 and
CD53 expression, and a down-regulation of CD5 and CD23 (Table 1). A fourth discrete
CD19+ B-cell subset with higher light-scatter characteristics and a PC phenotype - CD20
-,
DOI: 10.3324/haematol.2009.018689
6
CD38++
, CD27++
and cytoplasmic Ig (cyIg)+, with heterogeneous positivity for CD138
(57%±12% CD138- cells and 43%±12% CD138
+ cells) - was detected in all 106 HDs
analyzed.
Fluctuation of peripheral blood B-cell subsets according to age
In our large cohort of 106 HDs, naïve and memory B-lymphocytes were highly
represented, while immature B-lymphocytes and PC were minor populations (Table 1). No
correlation was found between age and percentages or absolute counts of total circulating
B-cells, immature B-lymphocytes or naïve B-lymphocytes (Figure 2A). In contrast,
statistically significant inverse correlations were found between age and both the
percentage and absolute count of circulating memory B-lymphocytes (n=106; R2$-0.22,
P$.05) and PC (n=106; R2$-0.27, P$.02). This also holds true when Ig heavy chain
isotype-specific subsets of PC (IgG+, IgA
+ and IgM
+) and memory B-lymphocytes (only for
IgG+ and IgM
+) were considered separately (Figure 2B). Our results indicate that
production of immature and naïve B-lymphocytes is not significantly affected by aging, in
contrast to what has been previously suggested by others.(12, 16)
On the contrary,
differentiation of naïve B-lymphocytes into memory B-lymphocytes and then PC, is clearly
reduced. These findings would confirm and extend on previous observations describing
alterations on B-cells consisting of a more restricted diversity.(12, 16)
In principle, this could
not be attributed to a lower ability for Ig class switch since we have reported here decreased
numbers of both non-switched IgM+/IgD
+, and switched IgG
+ memory B-lymphocytes with
aging.(13)
This age-related decrease in memory B lymphocyte counts could potentially be
due to a lower exposure to new Ag (associated with a less-exposed lifestyle) leading to a
more restricted memory B-cell repertoire or to an exhausted ability of memory B-
DOI: 10.3324/haematol.2009.018689
7
lymphocytes that have been triggered many folds along the lifespan of elderly people, to
generate expanded responses. The age-associated decrease in the number of circulating
IgA+, IgG
+, or IgM
+ PC was even more pronounced than that of memory B-lymphocytes,
suggesting the occurrence of lower humoral response rates in the elderly. In line with this
hypothesis, no significant correlation was found in our study between Ig heavy chain
isotype-specific circulating PC and their serum antibody counterpart (Supplementary
Figure S2).
Detailed characterization of circulating CD138-and CD138
+plasma cells
FACS-sorted CD138-CD20
-CD38
++ and CD138
+CD20
-CD38
++ cells showed a
typical PC cytology with no obvious morphological differences (Figure 1C). CD138+
PC
showed a greater staining index (SI) than CD138- PC for CD38 (22% increased SI; n=30,
P=.002), cyIg " and ! light chains (47% and 98% increased SI, respectively; n=6, P=.04),
CD27 (117% increased SI, n=12, P=.0004), and a lower SI for CD45 (24% decreased SI;
n=6, P=.004) (Figure 1D). In addition, CD138- PC, unlike CD138
+ PC, expressed weakly
CD20 and sIg (Figure 1D) consisting of sIgA+ (49%±12%), sIgG
+(13%±11%), sIgM
+sIgD
-
(18%±12%) and sIgM-sIgD
+ (5%±17%), but no sIgM
+/sIgD
+PC. After cell
permeabilization, 42%±14% cyIgA+, 31%±14% cyIgG
+ and 26%±10% cyIgM
+ PC were
identified (Figure 1B), with no differences in isotype distribution between CD138- and
CD138+ PC. The lower frequency of sIgG
+ vs. cyIgG
+ PC might be due to the weak sIg
expression that is not optimally detected by the anti-IgG Ab or a more mature sIgG-
phenotype of circulating PC. Although HLA-class II (including HLA-DR) expression was
heterogeneous and lower in circulating PC compared to PB B-lymphocytes (n=6, P".001),
a similar expression was found in CD138- vs. CD138
+ PC (Figure 1D). Unlike B-
DOI: 10.3324/haematol.2009.018689
8
lymphocytes, both CD138- and CD138
+ PC were CD43
+. Regarding homing receptors, both
CD138- and CD138
+ PC showed higher levels of %4 integrin (n=6, P".03), heterogeneously
lower amounts of CXCR4 (18.8±9.1% and 11.0±6.1%, respectively; n=6, P".0001), and
negativity for CD200 as compared to B-lymphocytes, which were constantly positive for
the later two markers (Figure 1D). CD138+ PC expressed lower levels of #7 integrin (n=6,
P=.02) and L-selectin/CD62L (n=5, P=.03) and higher levels of #1 integrin (n=6, P=.008)
than B-lymphocytes (Figure 1D). CCR10 was not expressed by circulating B-lymphocytes
while it was weakly positive on both CD138- and CD138
+ PC. Of note, high CCR10
expression was detected on the XG-1 and XG-10 myeloma cell lines or on in vitro
generated plasmablasts with the same anti-CCR10 mAb reagent (supplementary Figure
S3).(17, 18)
No significant difference in CCR10 expression was found among PC subsets
showing different Ig heavy chain isotypes (data not shown). Furthermore, both circulating
B-lymphocytes and CD138- and CD138
+ PC were constantly negative for VCAM1
(CD106), %5 integrin (CD49e), LFA-3 (CD58), and CD70, as well as for the CD56 and
CD117 markers, which are aberrantly expressed by malignant PC (data not shown).(19)
Based on KI-67 antibody, circulating B-lymphocytes were quiescent (1.2 %±0.8% KI-67+
cells) while circulating CD138- or CD138
+ PC displayed a highly-activated phenotype with
66.8%±29.7% and 76.2%±12.5% KI-67+ cells, respectively (n=11; P".00003, Figure 1D).
Further staining with Annexin-V showed that both CD138- and CD138
+ PC were fully
viable with only 6.8% and 7.7% of these cell subsets showing Annexin-V+ staining,
respectively.
Mei et al have suggested that the great majority of circulating PC could have a
mucosa origin, because they secrete mainly IgA (84%) and partially express CCR10.(3, 6)
DOI: 10.3324/haematol.2009.018689
9
We did not confirm these results, since only 40%-50% of all PB PC were IgA+ and CCR10
was very weakly expressed by circulating PC. This discrepancy is not due to a defect of the
anti-CCR10 mAb used (Supplementary Figure S3), but could be due to a difference in the
gating strategy to define PC and avoid contaminating cells. We used a gating on
CD19+CD38
high cells that comprise all and only cyIg" or cyIg! positive cells. Mei et al.
used two gating strategies, either CD19+CD27
high cells or cyIg
+ cells.
(3)
What is the origin and behavior of these circulating PC? Given mainly their HLA-
DR and CD45 expressions, they are generally thought to be plasmablasts newly-generated
in lymphoid organs. But HLA-DR and CD45 expressions are also characteristics of long-
living PC. A large fraction of CD138+
BM PC expresses HLA-class II (60%) or CD45
(65%).(2, 20)
PC, which are believed to be long-living based on murine models or by grafting
human PC in SCID mice (6)
, are also present in the spleen, MALT or lymph nodes. They
are located in APRIL-rich niches in the subepithelium(5)
, APRIL being an important PC
survival factor.(21)
The phenotype of these PC is close to that of circulating CD38++
CD138-
PC we presently reported. In human spleen, these PC are located outside the follicles,
express highly CD38, cyIg (45% cyIgM, 40% cyIgG and 15% cyIgA), and, unlike BM PC,
weakly sIg, CD20 and did not express CD138(1)
. They also express HLA-DR and CD45
and have mutated Ig genes.
The possibility that a fraction of circulating PC could be BM and/or lymphoid-
tissue-localized long living PC that are induced to re-circulate from their niche should be
considered. Such a recirculation of PC was hypothesized to explain the appearance of
circulating tetanus toxin-unrelated PC, 7 days after immunization of HDs with the toxin.(7)
If this mechanism occurs in case of tetanus toxin immunization, it may also occur in steady-
DOI: 10.3324/haematol.2009.018689
10
state conditions with newly-generated circulating plasmablasts competing with long living
PC. The activation status of the circulating PC (KI-67+) could indicate that they have been
induced to recirculate by local stimulation. Such a highly-regulated recirculation
mechanism has been demonstrated for murine HSC with circadian variations.(22)
These
circulating HSC can home to non-hematopoietic tissues for a time to exert immune
surveillance and may enter back the PB via lymphatics and thoracic duct.(23)
As human BM
PC use a stromal niche that is similar to that used by HSC (24)
and as the count of
circulating CD34+ cells in steady-state conditions is similar to that of circulating PC in
HDs, similar mechanisms could drive the circulation of HSC and PC.
DOI: 10.3324/haematol.2009.018689
11
Immature Naive Memory Plasma cells
PB Distribution*:
% from all PB B-cells
3.4%±2.4%
(1.8%-4.5%)
65%±12%
(57%-73%)
31%±12%
(22%-39%)
1.3%±1.4%
(0.5%-1.4%)
Absolute count(N. of cells/_L)
6±6
(2-9)
125±90
(63-156)
58±42
(28-68)
2.1± 2.1
(0.6-2.9)
Phenotypic profile**:
CD5 ++ -/+ - -
CD10 + - - -
CD19 + + + +d
CD20 + + + -
CD21 + + + -
CD22 + + + -
CD23 - -/++ - -
CD24 + +d + -
CD25 - - ++ -
CD27 - - + ++
CD34 - - - -
CD38 + -/+d -/+d ++
CD40 ++ ++ ++ +
CD43 - - - +
CD45 ++ ++ ++ +/++
CD53 + + ++ +d
CD80 - - - -
CD81 ++ + + +
CD86 - - - +
CD95 - - -/+ +
CD138 - - - -/+d
CCR6 + + + -
HLA-DR ++ ++ ++ +/++
sIgH ++(M&D)
+(M&D)
++(M&D or G or A)
+d
(M or D or G or A)
sIgL ++ + + +d
Table 1. Distribution and immunophenotypic profile of peripheral blood B-cell subsets.
*Results expressed as mean ± one standard deviation (p25-p75).
**Intensity of the staining expressed as -: negative, +d: dimly positive, +: positive, ++:
brightly positive.
DOI: 10.3324/haematol.2009.018689
12
Authorship and disclosures
AC and MPA performed the experiments, designed research, and wrote the paper. BP, CB,
AS, GF contributed in performing the experiments. NB, HJ contributed in writing the
paper. BK and AO designed research and wrote the paper. The authors reported no
potential conflicts of interest.
Acknowledgments
The authors would like to gratefully acknowledge Geneviève Fiol, Christophe Duperray,
Julia Almeida, Kirsten Fogd, and Jesus F. San Miguel.
DOI: 10.3324/haematol.2009.018689
13
FIGURE LEGENDS
FIGURE 1. Phenotype of peripheral blood B-cell subsets, and focus on circulating
plasma cells. Peripheral blood cells from healthy adult donors were analyzed by flow
cytometry. (A) Distribution of immature (green), naïve (pink), memory B-lymphocytes
(orange), and plasma cells (red), according to the expression of CD10, CD27 and CD38.
Cells were gated according to the strategy defined in the supplementary appendix (design
and methods section). (B) Cell phenotype was analyzed by gating on CD19+CD20
+CD38
-/+
- naïve and memory - B-lymphocytes (green), and CD19+CD20
-CD38
++ plasma cells
(blue). Histograms and dotplots show FACS labelings of cytoplasmic Ig (cyIg) - cyIgA,
cyIgG, and cyIgM - of one sample representative of 13. The contribution of each Ig isotype
is represented in the pie charts and numbers are the mean percentages ± one standard
deviation for the 13 healthy individuals. (C) CD20-CD38
++CD138
+ (a) and CD20
-
CD38++
CD138- (b) peripheral blood plasma cells from an adult healthy donor were FACS
sorted and stained with May-Grünwald-Giemsa (x1000 magnification). They showed
eccentrically nucleus, relatively abundant basophilic cytoplasm, and archoplasm. (D) Open
histograms show FACS labellings with anti-CD138, CD38, CD19, cytoplasmic Ig kappa or
lambda light chains (cyIg" or cyIg!), CD27, CD45, CD20, surface Ig heavy chains (sIg),
HLA class II, CD43, KI-67, %4 integrin (ITG%4), CXCR4, CD200, ITG#7, CD62L,
ITG#1, and CCR10 mAbs. Gray histograms display the corresponding negative control
mAbs. The cell phenotype was analyzed by gating on CD19+CD20
+ B-lymphocytes, and
both CD38++
CD138- and CD38
++CD138
+ PC. Data from one representative experiment is
shown. Numbers in panels indicate mean values of the staining indexes for each specific
DOI: 10.3324/haematol.2009.018689
14
mAb used or the percentage of positive cells, determined on between 3 to 30 different
healthy donors depending on the antibody used. *, # and § indicate that the values observed
are significantly (p$.05) different (paired Student t test) between B-lymphocytes and
CD138-
plasma cells (*), CD138- and CD138
+ plasma cells (#), and B-lymphocytes and
CD138+ plasma cells (§).
FIGURE 2. Age-related changes in circulating B-cell subsets and in the
immunoglobulin heavy chain isotype subsets of memory B-lymphocytes or plasma
cells. (A) Data plotted in each diagram represent correlation between the age of each
individual healthy donor and the percentage and absolute counts of total B-cells (panels A
and F, respectively), immature (panels B and G), naïve (panels C and H), and memory
(panels D and I) B-lymphocytes, as well as plasma cells (panels E and J). (B) Data plotted
in each diagram represent correlation between the age of each healthy individual donor and
the absolute count of memory B-lymphocytes and plasma cells expressing IgG (panels A
and D, respectively), IgM (panels B and E), IgA (panels C and F).
ONLINE SUPPLEMENTARY FIGURE S1. Expression of CD10, CD20 and CD38 on
immature, naïve B-cells and plasma cells. Distribution of the different subsets: immature
(!), naïve (!) B-lymphocytes and plasmablasts/plasma cells (!) in bone marrow and in
peripheral blood according to the expression of CD10, CD27 and CD38.
ONLINE SUPPLEMENTARY FIGURE S2. Correlation between serum
concentration of IgG, IgA and IgM antibodies and the percentage of the
corresponding Ig heavy chain isotype-specific memory B-cells. Correlation between the
concentration of IgG, IgA and IgM antibodies in the serum and the percentage of the
corresponding Ig heavy chain isotype-specific memory B-cells (A, B and C panels) and
DOI: 10.3324/haematol.2009.018689
15
plasma cells (D, E, and F). Serum IgM, IgG and IgA levels (mg/dL) were determined by
nephelometry.
ONLINE SUPPLEMENTARY FIGURE S3. Labeling of myeloma cells or in vitro
generated plasmablasts with anti-human CCR10 or anti-human IgA mAbs. The anti-
CCR10 mAb (clone 314305 from R&D systems) used was the same as that used by Mei et
al. This mAb strongly stained the CCR10+ XG1 human myeloma cell line, or in vitro
generated plasmablasts from healthy donors. The mAb to human IgA was also validated by
labelling the IgA+ XG-10 myeloma cell line after cell permeabilization.
DOI: 10.3324/haematol.2009.018689
21
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SUPPLEMENTARY APPENDIX
Design and Methods
Cell samples
EDTA-anticoagulated PB from a total of 106 adult healthy donors (63 men and 43 women;
age range: 20-82 years), were analyzed in this study, after informed consent was given by
each subject according to the Local Ethical Committees. Leukocyte concentrates from
healthy donors were obtained from the French Blood Center (Toulouse, France).
Antibodies. Antibodies (Abs) conjugated with pacific blue (PacB), anemonia majano cyan
(AmCyan), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll
protein/cyanin 5 (PerCP/Cy5), PerCP/Cy5.5, PE/cyanin 7 (PE/Cy7), allophycocyanin
(APC), alexafluor 700 (AF700), AF750, APC/Cy7, specific for human CD5 (clone
L17F12), CD9 (clone M-L13), CD10 (clone HI10a), CD19 (clone SJ25C1), CD21 (clone
B-ly4), CD22 (clone S-HCL-1), CD23 (clone EBVCS-5), CD24 (clone ML5), CD25 (clone
2A3), CD27 (clone L128), CD29 (#1 Integrin– ITG#1, clone MAR4), CD38 (clone HIT2
or HB7), CD40 (clone 5C3), CD43 (clone 1G10), CD45 (clones 2D1 and HI30), CD49d
(ITG%4, clone 9F10), CD49e (ITG%5, clone SAM1), CD53 (clone HI29), CD56 (N-CAM,
clone B159), CD62L (clone DREG-56), CD70 (clone Ki-24), CD80 (clone L307.4), CD81
(clone JS-81), CD86 (clone IT2.2), CD95 (clone DX2), CD106 (VCAM-1, clone 51-10C9),
CD138 (clone MI15), CD184 (CXCR4, clone 12G5), CCR6 (clone 11A9), HLA-DR (clone
L243), HLA-DR, DP, DQ (clone Tu39), ITG#7 (clone FIB504), Ig light chain lambda (Ig!,
clone JDC-12), Ig light chain kappa (Ig", clone TB 28-2), IgG (clone G18-145), IgM (clone
G20-127), and KI-67 (clone B56) were purchased from Becton/Dickinson (BD)
DOI: 10.3324/haematol.2009.018689
24
Biosciences (San Jose, CA); CD20 (clone B9E9), CD58 (LFA-3, clone AICD58) and
CD138 (clone B-A38) from Beckman Coulter (Fullerton, CA); CCR10 (clone 314305)
from R&D Systems (Minneapolis, MN); CD19 (clone HIB19), CD20 (clone 2H7), and
CD200 (clone OX104) were from eBiosciences (San Diego, CA); CD38 (clone HI72) from
ExBio (Vestec, Czech Republic); CD43 (clone TP1/36) from Immunostep (Salamanca,
Spain); IgM (polyclonal rabbit Ab), Ig! (polyclonal rabbit Ab), and Ig" (polyclonal rabbit
Ab) from Dako (Glostrup, Denmark); IgA (polyclonal goat antibody), IgG (polyclonal goat
Ab), and IgD (polyclonal goat Ab) from Southern Biotech (Birmingham, AL).
Immunophenotypic studies.
Erythrocyte-lysed whole PB samples or mononuclear cells obtained by Ficoll-hypaque
density gradient centrifugation were labeled with Abs conjugated with different
fluorochromes (2.106 cells in 100µL/test) as described.
(1) In some experiments, T and NK
cells were removed with anti-CD2 magnetic beads (DYNAL, dynabeads M-450 CD2 Pan
T) to enrich for B-cells. For intracellular staining of Ig or KI-67, cells were fixed and
permeabilized with the Cytofix/Cytoperm kit (BD Biosciences). B-cell subpopulations
were identified using a combination of 7-8 fluorochrome-conjugated Abs. The fluorescence
was analyzed with a FACSCanto II or a FACSAria flow cytometer, driven by the
FACSDiva 6.1 software (BD Biosciences). Data acquisition was performed using a two-
step procedure. Information from the total cellularity was first acquired using 5.104
cells/tube, and then, full data was specifically acquired for &2.105 cells showing CD19
and/or CD38 expression and low-to-intermediate sideward light scatter (SSC) values
(where B lymphocytes and PC are located). In multiparameter phenotyping, we used a first
gating on CD38high
cells because we have checked that CD38high
cells comprise all and only
DOI: 10.3324/haematol.2009.018689
25
cytoplasmic Ig PC in 10 PB samples. Multiparameter phenotyping of B lymphocytes was
performed on gated CD19+CD20
+ cells.
Data were analyzed with the Infinicyt 1.3 software (Cytognos SL, Salamanca, Spain). For
some mAbs, a bimodal fluorescence distribution was observed and the MFI was that of the
positive population. The fluorescence intensity of the cell populations was compared using
the staining index (SI) provided by the formula: (mean fluorescence intensity -MFI-
obtained from the given monoclonal Ab -mAb- minus MFI obtained with a control
mAb)/(2 times the standard deviation -SD- of the MFI obtained with the same control
mAb).(2)
Cell isolation and purification.
PC subpopulations, CD20-CD38
++CD138
- cells and CD20
-CD38
++CD138
+ cells, were
sorted with a FACSAria flow cytometry to perform cytospins. The purity of the sorted cells
was &90%. Cells were stained with May-Grünwald-Giemsa and cytology pictures were
acquired with a DM LB microscope (Leica, Wetzlar, Germany).
Statistical methods.
Mean values and their SD, median and range were calculated for continuous variables with
SPSS statistical software package (SPSS 10.1 Inc., Chicago, IL). Student-t test was used to
evaluate the statistical significance of differences observed between groups for paired and
unpaired variables. Correlation studies were performed using the Pearson test. P values
<.05 were considered to be associated with statistical significance.
DOI: 10.3324/haematol.2009.018689
26
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