Modulation of human B lymphocyte differentiation by therapeutic immunoglobulins: from protein to mRNA levels

doi:10.4236/oji.2011.13008

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Vol.1, No.3, 65-73 (2011)

doi:10.4236/oji.2011.13008

Open Journal of Immunology

Modulation of human B lymphocyte differentiation by

therapeutic immunoglobulins: from protein to mRNA

levels

Nathalie Dussault1, Nellie Dumont1, Sonia Néron1,2*

1Recherche et Développement, Héma-Québec, Québec, Canada; *Corresponding Author: sonia.neron@hema-quebec.qc.ca

2Département de Biochimie et de Microbiologie, Université Laval, Québec, Qc, Canada.

Received 17 August 2011; revised 18 October 2011; accepted 11 November 2011.

ABSTRACT

Several groups are investigating the mecha-

nisms of action of therapeutic immunoglobulins

(IVIg) in orde r to improve their use. In vitro mod-

els such as CD40-CD154 interaction are neces-

sary to study the physiological response of hu-

man B lymphocytes to IVIg. Human B lympho-

cytes treated with IVIg triggers a rapid phos-

phorylation (<1 h) of extracellular-regulatedkin-

ases 1 and 2 (ERK1/2), which subsequently re-

sults in increased differentiation and decreased

proliferation. However, the modulation of human

lymphocyte physiology by IVIg is a gradual and

cumulative process and requires long-term ex-

perimentation. Differentiation of human B lym-

phocytes into Ig-secreting cells can be evalu-

ated both at the transcription and translation

le vels . The secretion of immunoglobulins can be

assessed using ELISA or ELISPOTS whereas

expression of immunoglobulin genes can be

measured using semi-qua ntitative or quantitative

PCR methods. We hereby report a comparison

of these methods to explain how contradictory

observations towards IVIg effects could result

from their use. Our results indicate that ELISA

and ELISPOTS will provide consistent observa-

tions by opposition to real-time PCR quantifica-

tion. Besides, the reliability of each of these

methods remained dependent on the stimu lation

period as well as the preparation of cellular ex-

tract s or cell samples following IVIg-treatment.

Keywords: Human B Lymphocytes; CD40-CD154;

IVIg; Differentiation; Immunomodulation

1. INTRODUCTION

Therapeutic immunoglobulins are constituted of IgG

(≥98%) and are used in the treatment of several infla-

mmatory and autoimmune disorders in which IVIg have

been shown to reestablish homeostasis [1]. Following

IVIg injection, the concentration of IgG in the patient’s

serum increases by 5 to 10 mg/mL [2-4]. In many cases,

treatment with IVIg results in the reduction or disap-

pearance of pathologic autoimmune antibodies in the

patient’s serum for prolonged periods of time. Several

studies have proposed that anti-idiotypic antibodies,

present in IVIg, could interact with the B-cell receptor

(BCR). By doing so, IVIg modulate autoreactive B

lymphocytes [1]. According to this hypothesis, we show-

ed that IVIg interaction with B lymphocytes results in

phosphorylation of ERK1/2, which is downstream to

BCR cross-linking [5]. We also showed that IVIg can

directly modulate B lymphocytes by inducing secretion

of IgG reacting with self- and non-self antigens in cells

obtained from healthy individuals [6] as well as from

patient with systemic lupus erythematosus [7]. In vitro

models are important to study the mechanisms of action

of IVIg and several groups are using the CD40-CD154

interaction to delineate their effects on human B lym-

phocytes. However, all in vitro models using this inter-

action are not equal [8]. Variations in the level of CD40-

CD154 interactions can result in phenotypic and func-

tional differences in human B lymphocyte responses

[9,10] as well as in dendritic cells [11] and macrophages

[12]. Cell fate determination toward proliferation or dif-

ferentiation is directly proportional to the average num-

ber of CD154 molecules per B lymphocyte [9,13]. Es-

sentially, low levels of CD40 occupancy (500 to 1000

CD154 molecules per B cell) result in high Ig secretion

and low proliferation. Conversely, low Ig secretion and

high proliferation is observed when using a high level of

interaction (5000 to 10,000 CD154 molecules per B cell)

[9,13]. Moreover, sub-populations of human B lympho-

cytes will respond distinctively to variable intensity of

CD40 stimulation [9,13-15]. Therefore, models based on

in vitro CD40-activation of human B lymphocytes may

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

lead to very contrasting physiological effects, which are

as diverse as the nature of the ligands used to bind CD40

(reviewed in [8]). As a result and on the basis of our pre-

vious studies, the strength of IVIg modulation on B

lymphocytes will depend on the level of CD40-CD154

interaction [7].

For the present work, we have used low and high

levels of CD40-CD154 interaction and compared meth-

ods to assess the direct effect of IVIg on those B lym-

phocytes. We specifically targeted the differentiation

status by using ELISA and ELISPOT methods to deter-

mine the secretion rates and the frequency of secreting

cells and we used polymerase chain reaction (PCR),

semi-quantitative PCR and real-time PCR (Q-PCR) to

evaluate immunoglobulins’ expression levels.

2. MATERIALS AND METHODS

2.1. Intravenous Immunoglobulins

Commercial preparation of GAMUNEX® IVIg con-

taining 9% to 11% of protein and 160 to 240 mM gly-

cine, was obtained from Talecris (North Carolina, USA)

and albumin from bovine serum (BSA, Cohn fraction V,

Sigma-Aldrich Ltd, Oakville, ON, Canada) was prepared

at 10% in 10 mM phosphate buffered saline (PBS). IVIg

and BSA preparations were dialysed against 10 mM po-

tassium/sodium phosphate containing 136 mM NaCl

(PBS) (Gibco, Grand Island, NY, USA) and 40 mM gly

cine, pH4.5 (Sigma-Aldrich). After dialyse, IgG content

in IVIg was mainly monomeric (96% - 98%) and the

final concentration was 100 mg/ml [5]. BSA, which does

not interfere with B-cell proliferation and IgG secretion

[6], was used as a control for protein content.

2.2. Human Peripheral B Lymphocytes

This study has been reviewed and approved by Héma-

Québec’s Research Ethics Committee. Samples were

obtained from healthy individuals after informed consent.

Peripheral blood mononuclear cells (PBMNCs) were

prepared from leucocytes recovered from leucoreduction

system by density centrifugation over Ficoll-Paque (GE

Healthcare Bio-Sciences Inc., Baie d’Urfé, QC, Canada)

and stored frozen until B-cell purification as described

previously [16]. B lymphocytes were purified by nega-

tive selection using the Easy Sep CD19 cocktail, ac-

cording to the manufacturer’s instructions (Stem Cell

Technologies, Vancouver, BC, Canada) and were pure at

90% to 95% CD19+ cells, as determined by flow cy-

tometry.

2.3. CD40 Stimulation of B Lymphocytes

L4.5 cell line is a genetically modified L929 cell line

(CCL-1, American Type Culture Collection, Manassas,

VA) [17] expressing 21,000 ± 4000 CD154 molecules

per cell [9]. Purified B lymphocytes were seeded in Pri-

maria plates (BD Labware) at ~3.75 × 105 cells/mL in

the presence of γ-irradiated L4.5 cells expressing CD

154 [18]. L4.5 cells were seeded as to obtain ratios cor-

responding to high and low levels of CD40-stimulation,

3 - 5 or 20 - 25 B lymphocytes per L4.5 cell, respectively.

B lymphocytes were cultured in a medium based on

IMDM supplemented with 10% ultra low IgG FBS con-

taining 10 g/mL insulin, 5.5 g/mL transferrin, 6.7 ng/mL

sodium selenite, 100 μg/ml streptomycin and 100 U/ml

penicillin G (all from Invitrogen, Burlington, ON, Can-

ada), 50 U/mL IL-2, 25 U/mL IL-10 (PeproTech, Rocky

Hill, NJ, USA) and 100 U/mL IL-4 (R&D Systems,

Minneapolis, MN, USA). This complete IMDM medium

was used in all assays. B lymphocytes were stimulated

with high or low level of CD40-CD154 interaction for 9

days in the presence or absence of 10 mg/ml of IVIg.

When indicated, cells were cultured in the presence of

10 mg/mL of BSA or 4 mM glycine pH4.5 as controls

for IVIg-addition. Cultures were fed by replacing half of

the culture medium every 2 - 3 days, while L4.5 cells

were renewed every 4 - 5 days. Cell counts and viability

were evaluated in triplicates by trypan blue dye exclu-

sion using a hemacytometer. Cultured B lymphocytes

were always >96% CD19+ cells. Generation time (Tgen)

was calculated according to the formula: 1ln2



t2t1 21

ln 2[N]ln[N]tt



 and Tgen = 1/.

2.4. ELISA

For the determination of IgG and IgM secretion rate,

cells were harvested and washed 5 times to remove re-

sidual IVIg with PBS containing 2 g/L of glucose (PBS-

sglc). To validate that the secretion by the treated cells

was de novo protein synthesis, the washed cells were

incubated in complete IMDM medium for three hours

in the presence or absence of 15 μg/ml cycloheximide

(CHX). At this step these 3 h-supernatants were kept

and the cells were washed in PBS-glc and seeded at 1 -

2 × 106 cells/mL in IMDM medium alone for 18 to 22

hours. IgG and IgM concentrations were determined in

a standard ELISA using plastic-adsorbed goat antibod-

ies specific to human γ- and μ-chains and the bound

IgG or IgM were both revealed with peroxidase conju-

gated goat polyvalent anti-human Ig antibodies. All

antibodies were obtained from Jackson Laboratories

(Mississauga, Canada).

2.5. ELISPOTS

B lymphocytes, untreated or treated with IVIg, were

washed 5 times in PBS-glc, transferred in IMDM con-

taining 10% FBS and incubated for 3 hours with or

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

6767

without 15 µg/mL CHX. B lymphocytes were washed

and plated in 96-well Multiscreen IP sterile plate

(Milli-pore, Billerica, MA, USA) adsorbed with goat

anti-bodyies specific to human γ-chain following manu-

facturer’s instructions. B lymphocytes were added in

triplicates using serial dilutions varying from 20,000 to

156 cells/well, and incubated overnight at 37˚C in a

CO2-incubator. Plates were washed with 0.85% NaCl

and incubated with peroxidase-conjugated goat polyva-

lent anti-human Ig antibodies for 2 h at 37˚C in 10% CO2.

All antibodies were obtained from Jackson Laboratories.

Spots were revealed by incubation with TrueBlue TMB

Peroxidase Substrate (Mandel Scientific Cie Inc., Guel-

ph, Canada) for 30 minutes at room temperature. Mem-

branes were washed and dried overnight in the dark and

their numbers were determined using a microscope. The

frequency of secreting cells was determined by dividing

the number of spot units by the amount of seeded cells for

at least two successive dilutions.

2.6. Reverse Transcriptase-PCR and

Semi-Quantitative PCR

Purified B lymphocytes stimulated as described above,

in absence (CTL) or presence of 10 mg/ml BSA or 10

mg/ml IVIg, were washed 5 times to remove IVIg. RNA

extraction was performed using High Pure RNA iso-

lation kit (Roche diagnostics, Indianapolis, IN, USA)

according to the manufacturer’s instructions. RNA was

treated with DNAse Amp grade (Invitrogen) and first-

strand cDNA was synthesized using M-MLV RT (Invi-

trogen). PCR was performed on PCRExpress (Thermo-

Hybaid, Ashford, UK) using AmpliTaq Gold (Applied

Biosystems, Foster City, CA, USA) following manufac-

turer’s instructions. Amplification of gamma regions of

IgG1, IgG2, IgG3, IgG4 [19] and IgG1-S, IgG2-4 [20], was

done using the primer combinations listed in Tab l e 1 , at

95˚C for 5 minutes followed by 35-cycles at 95˚C for 15

seconds and 60˚C for 30 seconds, with a final elongation

step at 72˚C for 6 minutes. Semi-quantitative PCR was

performed using the same method, but using an initial

denaturation at 95˚C for 5 minutes, followed by 15, 20, 25,

30 and 35 PCR cycles. All PCR products were separated

by electrophoresis on a 2% agarose gel (Invitrogen).

2.7. Quantitative Real-Time-PCR

Q-PCR was performed on Stratagene Mx3005P QPCR

system using PerfeCta SYBR® Green FastMixTM

(Quanta Bioscience Gaithersburg, MD, USA) and master

Mix 1 following manufacturer’s instructions. Amplifica-

tion of gamma region of IgG, IgG1, IgG2, IgG3 and IgG4,

as well as glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) [21,22] or 18S ribosomal RNA [20] as en-

Table 1. Primers for PCR and Q-PCR.

F1CAAGGACTACTTCCCCGAAC

IgG RTCTTGTCCACCTTGGTGTTTG 2002

FGCATGTACTAGTTTTGTCACAAGATTTGGG

IgG1RTCCACCAAGGGCCCATCG 300

F CTCGACACTAGTTTTGCCGCTCAACTGTCTT

IgG2RTCCACCAAGGGCCCATCG 300

F TGTGTGACTAGTGTCACCAAGTGGGGTTTT

IgG3RTCCACCAAGGGCCCATCG 300

F GCATGAACTAGTTGGGGGACCATATTTGGA

IgG4RGCTTCCACCAAGGGCCCATC 300

FCATCTCCAAAGCCAAAGG

IgG1-S RATGTCGCTGGGATAGAAG 150

FCATCTCCAAAGCCAAAGG

IgG2-4 RATGTCGCTGGGGTAGAAG 150

FCGAGATCCCTCCAAAATCAA

GAPD

H RGTCTTCTGGGTGGCAGTGAT 300

FAGTCCCTGCCCTTTGTACACA

18S

rRNA RGATCCGAGGGCCTCACTAAAC 68

1Forward (F); Reverse (R) 2Amplicon (nts).

dogenous control genes, was done using the primers

listed in Ta ble 1. Amplification of gamma region of IgG

and GAPDH yielded amplicons of ~300 nt. When indi-

cated quantification of GAPDH or 18S rRNA was per-

formed for each sample; allowing normalization of sam-

ples as previously described [23]. Dissociation curve

analysis was performed to obtain the amplification of a

single PCR product. Quantification of the transcripts was

carried out with the software Mx3005P version 2.02 (In-

vitrogen) using the comparative threshold cycle 2–ΔΔCt

method.

2.8. Statistical Analysis

When indicated, the mean values ± standard devia-

tions or standard error of the mean (SEM) were calcu-

lated, data distribution was evaluated using the Shapiro-

Wilk test and analysis of variance was done using the

F-test. Thereafter, statistical significance between tests

and controls was determined using Mann-Whitney U-

test or two-sided Student’s paired t-test.

3. RESULTS

3.1. IVIg and B Lymphocyte Proliferation

Human B lymphocytes, isolated from 6 independent

samples were stimulated with high or low level of CD-

154 and cultured for 9 days, in the presence or absence

of 10 mg/ml IVIg. IVIg were added from the start and

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

maintained in medium renewal for the duration of the

culture (Figure 1). Total expansion was determined over

the 9-day culture period (Figure 1(a)) and Tgen was de-

termined for cells receiving high (Figure 1(b)) and low

(Figure 1(c)) CD40-CD154 interaction within the expo-

nential growth phase, namely between days 4 and 9. As

previously reported [9,13], Tgen mean value appeared

higher for cells submitted to low (54 ± 16 hours) than for

those submitted to high (43 ± 15 hours) CD154 interac-

tion. However, this tendency was not statistically differ-

ent (p = 0.267). In both cases, addition of IVIg signify-

cantly increased the Tgen by about 20% when compared

to untreated cells: 52 ± 17 hours and 63 ± 23 hours for

cells submitted to high and low level of CD154 interac-

tion, respectively. Worthy of note, there was no differ-

ence in growth rate during the initiation phase, from day

0 to 4, whether the cells were submitted to high or low

CD40-stimulation and in presence or absence of IVIg.

Essentially, these 6 independent experiments showed

that significant IVIg reduction of B lymphocyte prolif-

eration was observed only after a 9-day IVIg treatment

in cells maintained in exponential growth.

3.2. Residual IgG in B Lymphocyte Sample

Following IVIg Treatment

In vitro IVIg treatment of B lymphocytes using 10

mg/ml or higher concentrations is highly problematic for

the determination of IVIg effects on IgG secretion.

However, we previously reported [6] that such measure-

ments can be done when using cells that have been

washed extensively and seeded back to secrete in abs-

ence of IVIg for a short period (<24 h). To illustrate how

important and efficient was the repeated washings of B

lymphocytes, we have measured the residual IgG content

in washing supernatants of IVIg treated-cells (Figure 2).

A minimum of 5 washing steps were needed to decrease

the concentration of IgG (IVIg) from 10 mg/ml to less

than 500 ng/mL of IgG. These successive washes also

resulted in the recovery of a final amount of 2 - 4 × 106

cells, representing less than 30% of the cells collected at

the end of the culture period. Therefore, when 18 to 21

hours-secretion periods were evaluated, the IgG content

coming from IVIg could correspond to a residual con-

centration of 125 to 250 ng per 106 cells.

3.3. IVIg Increase Differentiation into

IgG-Secreting Cells

As described in Figure 1, cultured B lymphocytes

were washed and treated for 3 hours in the presence or

absence of CHX. Then, these 3 h-treated B lymphocytes

were washed once more and seeded back in cultureme-

dium for 18 hours to 21 hours. This two-step incubation

enables us to discriminate between de novo synthesis

(a)

(b)

(c)

Human B lymphocytes (>95% CD19+ cells) stimulated for 9 days with

high (square symbols) and low levels (triangle symbols) of CD154

interaction in presence (IVIg; empty symbols) of 10 mg/ml IVIg or

with an identical volume of 40mM glycine buffer (CTL; filled sym-

bols). Error bars for triplicates, can be smaller than symbols. These

results are representative of 6 independent experiments. Generation

time values (Tgen) for B lymphocytes exposed to high (b) and low (c)

level of CD154 are showed for 6 independent samples. P values for

Tgen comparing IVIg-treated and untreated cells were determined using

two-sided Student’s paired t-test and correlation coefficient were r =

0.968 (B) and r = 0.982 (c). In B and C, each histogram pattern stands

for one sample. For example filled histograms in B and C are the same

sample ± IVIg in both conditions.

Figure 1. IVIg inhibit B lymphocyte proliferation.

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

6969

of IgG following IVIg treatment and releasing of IVIg,

which could occur following spontaneous internalization

[24]. IgG and IgM secretion rates were established for 6

independent samples following incubation in the pres-

ence or absence of IVIg and compared to CHX-treated

cells (Figures 3(a)-(b)). This CHX-treatment inhibited

IgG and IgM secretion by more than 80% for at least 18

to 21 hours (Figures 3(a)-(b)) and inhibition of protein

synthesis was already noticeable after 3 hours (Figure

3(c)). As suggested by the large SEM (Figure 3), such in

vitro CD40-activation is characterized by inter-individ-

ual variability in the human B lymphocytes response

[7,9,15,25]. Still, the comparison of 6 experiments using

two-sided Student’s paired t-test allowed us to highlight

significant differences.

Figure 2. Removal of 99% of residual IgG in IVIg treated cells.

B lymphocytes have been stimulated with high or low CD154

interaction, as described in Figure 1, in the presence of 10

mg/mL IVIg for 9 days. A total number of 16 - 20 × 106 cells

were washed with PBS-glc 5 times. IgG content was measured

in each washing supernatants by ELISA. Data are presented as

the mean ± SD of 3 independent experiments.

Human B lymphocytes submitted to high levels of

CD40-CD154 interaction and treated with IVIg were

secreting 18 to 208 ng of IgG/h/106 cells (mean ± SD; 97

± 70) while control cells were secreting 6 to 89 ng of

(a) (b)

Figure 3. IVIg-increased de novo IgG secretion. B lymphocytes were stimulated as described in Figure 1. On day 9, cells were

washed 5 times and incubated 3 hours in the absence or presence of 15 µg/mL cycloheximide (CHX). Cells were washed and then

seeded back in IMDM alone for a supplemental 18 to 20 h. IgG and IgM contents were determined in these supernatants ((a) and (b))

and in the 3 h-supernatant (c). The frequency of IgG-secreting cells was determined by a standard ELISPOT (d). Data presented in A

and B are the mean ± SEM of six independent experiments while results presented in (c) are the mean ± SEM of four independent

experiments. The IVIg increase of IgG secretion in B lymphocytes submitted to high (p = 0.030) and low (p = 0.09) levels of

CD40-CD154 interaction is significant according to paired t-test while no significant difference was observed for IgM secretion ((a)

and (b)). The IVIg induced IgG secretion during CHX-treatment is significant for cells submitted to high level of CD40-CD154 in-

teraction (U = 2,309; p = 0.0209).

Openly accessible at

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

IgG/h/106 cells (mean ± SD; 50 ± 35). Thus, high levels

of CD40-CD154 interaction clearly led to a statistically

significant IVIg-increase of IgG secretion (p = 0.0300).

On the other hand, human B lymphocytes submitted to

low stimulation in the presence of IVIg were secreting

111 to 2951 ng of IgG/h/106 cells (mean ± SD; 1042 ±

1014) while untreated cells were secreting 65 to 1550 ng

of IgG/h/106 cells (mean ± SD; 681 ± 544). Such an in-

crease of IgG secretion (p = 0.09), namely about 1.5-fold,

corresponds to a supplemental secretion of 362 ± 513 ng

of IgG/h/106 cells. In contrast, IVIg did not modulate de

novo IgM secretion by B lymphocytes submitted to low

and high level of CD40-CD154 interaction (Figur e 3(b)).

IgG content in the supernatant from CHX-treated cells

in 4 independent experiments was also assessed (Figure

3(c)). IVIg increased IgG secretion by 1.8-fold and 4.6-

fold for cells submitted to low and high level of CD40-

CD154 interaction, respectively. We have also measured

IgG de novo synthesis by ELISPOT using CHX treated-

cells (Figure 3(d)). On day 9, the frequency of IgG se-

creting cells detected by ELISPOT assay was very low

(<3%), nonetheless IVIg still appeared to slightly in-

crease the frequency of IgG-secreting cells.

We have previously shown that IVIg-treatment of

human B lymphocytes increases IgG secretion in long-

term culture, namely 14 days [5-7]. These results further

substantiate that IVIg can increase the de novo IgG se-

cretion in CD40-activated B lymphocytes after a 9-day

treatment (Figure 1).

3.4. IVIg Modulation and mRNA Synthesis

B lymphocytes were cultured for 9 days, as above

(Figure 1), in the presence or absence of IVIg. In this

case, controls were treated with PBS-40 mM glycine pH

4.5 (CTL) and BSA as indicated methods. RT-PCR was

used to verify whether all 5 sets of primers used effi-

ciently detected gamma chains as well as subclasses

such as IgG1, IgG2 and IgG3 and IgG4 (Figures 4(a) and

(b)). Except for the primers used to detect IgG3, all sets

of primers used were reliable to detect the various IgG

mRNA from controls cells as well as cells treated with

BSA or IVIg. IgG3 mRNA was however detected in

some samples but showed no increase in IVIg-treated

cells (data not shown). For the set of primers used in to

detect IgG1 and IgG2 (Figure 4 (b)), these analyses, whi-

ch have been done up to 4 times on 3 independent sam-

ples, showed an increase in IgG mRNA content follow-

ing IVIg treatment. However, these observations were

not consistent with the other sets of primers used target-

ing the same mRNA.

Using semi-quantitative PCR, we evaluated whether

IVIg modulation of IgG transcription can be detected

(Figure 4 (c)). In this case, the two sets of primers used

in panel A target IgG1 (IgG1-S) and IgG2, IgG3 and IgG4

(IgG2-4) [20]. Although the quantitative aspect was im-

proved when compared to the RT-PCR experiments,

there was no increase in the IgG mRNA levels following

IVIg treatment when compared to untreated cells. BSA-

treated cells displayed lower expression levels of IgG1

mRNA than untreated cells or IVIg-treated cells. These

results did not reveal any significant effect of IVIg and

were contradictory with the observed increased of IgG

protein, as determined by ELISA, for the same experi-

ment and samples. For example, panel A and B corre-

spond to a culture where IgG secretion was increased by

2-fold in IVIg treated cells when compared to BSA-

treated cells and untreated cells, which were giving iden-

tical IgG secretion.

Q-PCR analyses were performed to compare the

mRNA expression of untreated cells and BSA-treated

cells to IVIg-treated cells (Figure 5). Relative IgG

mRNA content varied depending on the housekeeping

gene used and depending on whether IVIg-cells were

compared to untreated or BSA-treated cells. The twofold

increase, which is usually considered as a significant

difference in Q-PCR analysis, was not observed when

IVIg conditions were compared to untreated cells. Once

again, IVIg appeared to significantly increase IgG1 and

IgG2 expression when compared to the BSA condition.

Overall, neither semi-quantitative PCR nor Q-PCR

detected an increased IgG secretion in human B lym-

phocytes with IVIg. Furthermore, these experiments

carried out using 3 independent samples were fluctuating

depending on the samples used.

(a) (b) (c)

Figure 4. IgG mRNA is not influenced by IVIg. B lymphocytes were stimulated for 9 days with low levels of CD40-CD154 interac-

tion in the presence or absence of 10 mg/mL bovine serum albumin (BSA) or 10 mg/mL IVIg (IVIg) both dialyzed in 40 mM glycine

pH 4.5. For controls, 10% (v/v) 40 mM glycine pH4.5 was added in the culture medium (CTL). IgG mRNA levels were evaluated

using PCR method ((a) and (b)) and semi-quantitative PCR (c) with primers (Table 1) specifically targeting IgG, IgG1-s, IgG2-4 , IgG1,

IgG2 IgG3 and IgG4 gamma chains, and GAPDH, as indicated at the top of the panels.

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

7171

(a) (b)

Figure 5. Q-PCR does not correlate with IVIg modulation of IgG. B lymphocytes were stimulated as above (Figure 4). In (a), two

housekeeping genes, GAPDH (filled histograms) and 18S rRNA (Shaded histograms), relative expression values are obtained by

comparing IVIg-treated cells to controls cells. In (b), relative values are for IVIg-treated cells compared to control (CTL; filled histo-

grams) or BSA-treated cells (BSA; hatched histograms) using GAPDH as a reference gene.

4. Discussion

We have previously shown that IVIg can have direct

effects on B lymphocytes [5-7]. Essentially, IVIg modu-

lation of human B lymphocytes translates into a diminu-

tion of proliferation concomitant to an augmentation of

Ig secretion. Such an increase in the secretion of human

IgG is caused by de novo synthesis, which has been

shown using metabolic labeling with 35S-methionine [6].

On the other hand, we have also highlighted that varia-

tion in the intensity of CD40-CD154 interaction can

strongly modulate the evolution of human blood B lym-

phocytes and alter the outcome of naïve and memory

populations [8,9,13] as well as their modulation by IVIg

[7]. Therefore, interpretation of IVIg-modulation of hu-

man B lymphocytes, using such in vitro models, depends

on the nature and strength of CD40 interaction and

might be a source of discrepancy between studies.

In this study, we used high and low level of CD40-

stimulation in a cell-to-cell stimulus provided by CD

154+ L cells [17] and human B lymphocytes treated with

10 mg/mL of IVIg as previously done [7]. As expected,

proliferation was delayed in IVIg-treated cells and dif-

ferentiation was down-regulated and these changes were

also dependent on the level of CD40 stimulation.

The study of IVIg modulation of human B lympho-

cyte differentiation is limited by the in vitro treatment

because IVIg interfere with the measurements of IgG

secretion in the IVIg-treated cells [20,26]. To minimize

the contribution of IVIg in IgG measurements, we con-

stantly perform extensive washing of the treated cells

[5-7]. We show that these procedures effectively remove

99.9% of residual IgG, which is less than 500 ng/mL. On

the other hand, the extensive washings induce the loss of

60% - 70% of the cells during centrifugation, which is a

drawback when the amount of B lymphocytes is some-

what limited. Moreover, IVIg treatment not only pre-

cludes direct IgG measurements in culture supernatants,

but may also have potential interferences caused by its

high protein content, as recently reported for prolifera-

tion assays based on BrdU incorporation [27]. Therefore,

extensive washings should be done even if the assays are

not related to IgG measurements in order to avoid un-

wanted interferences.

To support the measurements of enhanced differe-

ntiation in IVIg-treated cells, we further emphasize on

de novo synthesis by using protein synthesis inhibitor

[28] in combination to ELISA or ELISPOT assays. The

cycloheximide treatment confirmed that IVIg induction

of IgG secretion and enhancement of IgG secreting cells

are mainly caused by novel synthesis.

In this study, we also investigated the effect of IVIg

on mRNA levels using PCR, semi-quantitative PCR and

Q-PCR as previously done [20]. All analyses were done

after 9 days of culture in the presence or absence of IVIg,

in conditions where human B lymphocytes exhibit gen-

eration times (Tgen) ranging from 30 to 70 hours [15].

Although we have used several sets of primers amplify-

ing any gamma chain as well as primers specific to each

gamma 1 to gamma 4 subclasses, our results were in-

conclusive mostly because not reproducible from one

samples to another. Worthy of note, such Q-PCR analy-

sis is adequate to monitor the 10-fold increase in IgG

secretion in B lymphocytes stimulated with low com-

pared to high CD40-CD154 interaction (data not shown).

The use of BSA as a control for IVIg addition is once

more questionable [7]. Semi-quantitative analyses showed

lower expression levels of IgG1 mRNA in BSA-treated

cells compared to untreated cells. Similar results were

obtained with Q-PCR analyses where BSA inhibits IgG

mRNA expression when compared to untreated cells

(data not shown). Besides, we observed a down regula-

tion of GAPDH mRNA expression in the BSA-treated

cells. GAPDH is considered as a protein content control

by many investigators. Therefore, when comparing IVIg-

treated cells to BSA-treated cells, increase mRNA ex-

pression could be incorrectly interpreted as a relative

higher expression for IVIg-treated cells. Those results

N. Dussault et al. / Open Journal of Immunology 1 (2011) 6 5-73

confirm previous studies showing that BSA have proper-

ties that make it unsuitable as a placebo [29].

On the other hand, the use of Q-PCR to monitor IVIg

modulation of IgG secretion is questionable, particularly

since a maximum of twofold increase secretion is often

observed. Besides, these twofold changes are not valid in

Q-PCR determination. In fact, the choice of reference

“housekeeping” genes can greatly influence the nor-

malization results and can vary from one type of cells to

another. As example, the choice of 18S rRNA to monitor

IVIg treatment [20], which reliability may vary from one

tissue to another [21,22,30] and is particularly inconsis-

tant in T lymphocytes [31]. Our Q-PCR analyses show a

maximum increase of 1.6-fold in IgG mRNA expression

following IVIg treatment, which is not considered sig-

nificant for this method. In a certain point of view, these

results are contradictory with the ELISA conclusion but

are in agreement with those of Heidt and collaborators

[20] stating that IVIg had no effect on IgG secretion ac-

cording Q-PCR analysis of mRNA levels.

To avoid IVIg interferences, the quantification of IgM

secretion have been previously used to monitor IVIg

influence on the immunoglobulin production in common

variable immunodeficiency, which was quite significant

[26]. However, IgM secretion is restricted to naïve and

memory subsets of B lymphocytes and is not representa-

tive of switched memory B lymphocytes. Moreover, our

results indicated that IVIg effects on IgM secretion was

less important and not significant compared to that on

IgG synthesis. Therefore, the method described here re-

presents an acceptable option to monitor the direct effect

of IVIg on B lymphocytes differentiation. Furthermore,

variations in the nature and intensity of CD40 binding

during in vitro activation of human B lymphocytes will

result in the emergence of distinct B-cell populations [9],

which might in turn give rise to differential responses to

IVIg [7].

In conclusion, by delimiting the strength and weakness

in ELISA and PCR methods used to monitor IVIg effects

on human B lymphocytes, this study intend to contribute

to a better understanding of the mechanisms of action of

IVIg from the protein to the mRNA level.

5. ACKNOWLEDGEMENTS

We thank all the volunteers who participated in this study and Clau-

dine Côté Inf. for scheduling blood sample collection. We are grateful

to Marc Cloutier Ph.D. for excellent critical review and manuscript

editing.

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