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

Publisher's Disclaimer.E-publishing ahead of print is increasingly important for the rapid dissemination of science.Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts thathave completed a regular peer review and have been accepted for publication. E-publishingof this PDF file has been approved by the authors. After having E-published Ahead of Print,manuscripts will then undergo technical and English editing, typesetting, proof correction andbe presented for the authors' final approval; the final version of the manuscript will then appe-ar in print on a regular issue of the journal. All legal disclaimers that apply to the journal alsopertain to this production process.

Haematologica (pISSN: 0390-6078, eISSN: 1592-8721, NLM ID: 0417435, www.haemato-logica.org) publishes peer-reviewed papers across all areas of experimental and clinicalhematology. The journal is owned by the Ferrata Storti Foundation, a non-profit organiza-tion, and serves the scientific community with strict adherence to the principles of openaccess publishing (www.doaj.org). In addition, the journal makes every paper publishedimmediately available in PubMed Central (PMC), the US National Institutes of Health (NIH)free digital archive of biomedical and life sciences journal literature.

Official Organ of the European Hematology AssociationPublished by the Ferrata Storti Foundation, Pavia, Italy

www.haematologica.org

Early Release Paper

Support Haematologica and Open Access Publishing by becoming a member of the European Hematology Association (EHA)and enjoying the benefits of this membership, which include free participation in the online CME program

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

16

DOI: 10.3324/haematol.2009.018689

17

DOI: 10.3324/haematol.2009.018689

18

DOI: 10.3324/haematol.2009.018689

19

DOI: 10.3324/haematol.2009.018689

20

DOI: 10.3324/haematol.2009.018689

21

REFERENCES

1. Stuart GT, David MT. Memory B cells: effectors of long-lived immune responses.

Eur J Immunol, 2009:2065-75.

2. Medina F, Segundo C, Campos-Caro A, Gonzalez-Garcia I, Brieva JA. The

heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals

graded stages of increasing maturity, but local profiles of adhesion molecule expression.

Blood. 2002 Mar 15;99(6):2154-61.

3. Mei HE, Yoshida T, Sime W, Hiepe F, Thiele K, Manz RA, et al. Blood-borne

human plasma cells in steady state are derived from mucosal immune responses. Blood.

2009 Mar 12;113(11):2461-9.

4. Costes V, Magen V, Legouffe E, Durand L, Baldet P, Rossi JF, et al. The Mi15

monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in

paraffin-embedded bone marrow biopsy specimens. Hum Pathol. 1999 Dec;30(12):1405-

11.

5. Mohr E, Serre K, Manz RA, Cunningham AF, Khan M, Hardie DL, et al. Dendritic

cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node

microenvironments where plasmablasts mature. J Immunol. 2009 Feb 15;182(4):2113-23.

6. Radbruch A, Muehlinghaus G, Luger EO, Inamine A, Smith KG, Dorner T, et al.

Competence and competition: the challenge of becoming a long-lived plasma cell. Nat Rev

Immunol. 2006 Oct;6(10):741-50.

7. Odendahl M, Mei H, Hoyer BF, Jacobi AM, Hansen A, Muehlinghaus G, et al.

Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma

cells in a secondary immune response. Blood. 2005 Feb 15;105(4):1614-21.

8. Marie-Cardine A, Divay F, Dutot I, Green A, Perdrix A, Boyer O, et al. Transitional

B cells in humans: characterization and insight from B lymphocyte reconstitution after

hematopoietic stem cell transplantation. Clin Immunol. 2008 Apr;127(1):14-25.

9. Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, Lipsky PE. Identification

and characterization of circulating human transitional B cells. Blood. 2005 Jun

1;105(11):4390-8.

10. Palanichamy A, Barnard J, Zheng B, Owen T, Quach T, Wei C, et al. Novel human

transitional B cell populations revealed by B cell depletion therapy. J Immunol. 2009 May

15;182(10):5982-93.

11. Lee J, Kuchen S, Fischer R, Chang S, Lipsky PE. Identification and characterization

of a human CD5+ pre-naive B cell population. J Immunol. 2009 Apr 1;182(7):4116-26.

12. Siegrist CA, Aspinall R. B-cell responses to vaccination at the extremes of age. Nat

Rev Immunol. 2009 Mar;9(3):185-94.

13. Frasca D, Landin AM, Riley RL, Blomberg BB. Mechanisms for decreased function

of B cells in aged mice and humans. J Immunol. 2008 Mar 1;180(5):2741-6.

14. LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood.

2008 Sep 1;112(5):1570-80.

15. Shearer WT, Rosenblatt HM, Gelman RS, Oyomopito R, Plaeger S, Stiehm ER, et

al. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric

AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol. 2003 Nov;112(5):973-

80.

DOI: 10.3324/haematol.2009.018689

22

16. Listi F, Candore G, Modica MA, Russo M, Di Lorenzo G, Esposito-Pellitteri M, et

al. A study of serum immunoglobulin levels in elderly persons that provides new insights

into B cell immunosenescence. Annals of the New York Academy of Sciences. 2006

Nov;1089:487-95.

17. Zhang XG, Gaillard JP, Robillard N, Lu ZY, Gu ZJ, Jourdan M, et al. Reproducible

obtaining of human myeloma cell lines as a model for tumor stem cell study in human

multiple myeloma. Blood. 1994 Jun 15;83(12):3654-63.

18. Jourdan M, Caraux A, De Vos J, Fiol G, Larroque M, Cognot C, et al. An in vitro

model of differentiation of memory B cells into plasmablasts and plasma cells including

detailed phenotypic and molecular characterization. Blood. 2009 (ahead of print).

19. Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, et al.

Report of the European Myeloma Network on multiparametric flow cytometry in multiple

myeloma and related disorders. Haematologica. 2008 Mar;93(3):431-8.

20. Pellat-Deceunynck C, Bataille R. Normal and malignant human plasma cells:

proliferation, differentiation, and expansions in relation to CD45 expression. Blood cells,

molecules & diseases. 2004 Mar-Apr;32(2):293-301.

21. Moreaux J, Sprynski AC, Dillon SR, Mahtouk K, Jourdan M, Ythier A, et al. April

and Taci Interact with Syndecan-1 on the Surface of Multiple Myeloma Cells to Form an

Essential Survival Loop. Eur J Haematol. 2009 Mar 30.

22. Mendez-Ferrer S, Lucas D, Battista M, Frenette PS. Haematopoietic stem cell

release is regulated by circadian oscillations. Nature. 2008 Mar 27;452(7186):442-7.

23. Massberg S, Schaerli P, Knezevic-Maramica I, Kollnberger M, Tubo N, Moseman

EA, et al. Immunosurveillance by hematopoietic progenitor cells trafficking through blood,

lymph, and peripheral tissues. Cell. 2007 Nov 30;131(5):994-1008.

24. Sugiyama T, Kohara H, Noda M, Nagasawa T. Maintenance of the hematopoietic

stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell

niches. Immunity. 2006 Dec;25(6):977-88.

DOI: 10.3324/haematol.2009.018689

23

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

References

1. Ocqueteau M, Orfao A, Almeida J, Blade J, Gonzalez M, Garcia-Sanz R, et al.

Immunophenotypic characterization of plasma cells from monoclonal gammopathy of

undetermined significance patients. Implications for the differential diagnosis between

MGUS and multiple myeloma. Am J Pathol. 1998 Jun;152(6):1655-65.

2. Maecker HT, Frey T, Nomura LE, Trotter J. Selecting fluorochrome conjugates for

maximum sensitivity. Cytometry A. 2004 Dec;62(2):169-73.