Impaired Ig class switch in mice deficient for the X-linked lymphoproliferative disease gene Sap

doi:10.1182/blood-2004-07-2731Prepublished online June 7, 2005;

Tavtigian, Zhao-Qi Wang, Sylvain Latour and Luo YinUmaima Al-Alem, Cuiling Li, Nathalie Forey, Francis Relouzat, Marie-Claude Fondaneche, Sean V lymphoproliferative disease gene sapImpaired Ig class switch in mice deficient for the X-linked

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1

Immunobiology

Impaired Ig Class Switch in Mice Deficient for the X-Linked

Lymphoproliferative Disease Gene sap†

Umaima Al-Alem1,2, Cuiling Li1, Nathalie Forey1, Francis Relouzat3, Marie-Claude Fondanèche3,

Sean V. Tavtigian1, Zhao-Qi Wang1, Sylvain Latour3‡ and Luo Yin1

1International Agency for Research on Cancer, Lyon, France2Division of Pulmonary, Allergy, and Critical Care Medicine, Columbia University,

New York, USA3Unité INSERM 429, Hôpital Necker-Enfants Malades, Paris, France

†This work is supported in part by grants from the Association for International Cancer Research,

Association pour la Recherche sur le Cancer, GIS-Institut des Maladies Rares, Institut Nationale de

la Santé et de la Recherche Médicale, and the Swiss Federal Office of Public Health.‡S.L. is a scientist from the Centre National de la Recherche Scientifique (France).

Correspondence should be addressed to:

Dr Luo Yin or Dr Sylvain Latour

Unité INSERM 429, Hôpital Necker Enfants-Malades

149, rue de Sèvres, 75015 Paris, France

Phone: +33 (0)1 44 49 50 51; Fax: +33 (0)1 42 73 06 40; E-mail: [email protected]

Short running title: Impaired Ig class switch in sap-deficient mice

Word counts: total text, 3500; abstract, 136

Blood First Edition Paper, prepublished online June 7, 2005; DOI 10.1182/blood-2004-07-2731

Copyright © 2005 American Society of Hematology




2

Abstract

X-linked lymphoproliferative disease is characterized by abnormal immune responses to Epstein-

Barr virus attributed to inactivating mutations of the SAP gene. Previous studies showed IgE

deficiency and low serum IgG levels in sap-deficient mice before and after viral infections, which

are associated with impaired CD4+ T helper function. In the present work, we find that SAP is

expressed in B cells and this expression is down-regulated after stimulation with LPS and IL-4. We

demonstrate that B cells from sap-deficient mice exhibit reduced IgG and IgA production in vitro.

This impairment correlates with decreased circular transcript levels of Iα, Iγ2a, Iγ2b, and Iγ3 after

stimulation, which indicate a defective Ig switch recombination in sap-deficient B cells. While XLP

is believed to cause defects in T, NKT, and NK cells, our results indicate that B cells are also

affected.




3

Introduction

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency with variable

phenotypes often associated with Epstein-Barr virus (EBV) infection. The most frequent clinical

manifestations of XLP are fatal or severe infectious mononucleosis, hypo- or agammaglobulinemia,

and non-Hodgkin lymphoma of Burkitt type.1,2 It has been demonstrated that inactivation or

dysfunction of SAP (also called SH2D1A or DSHP) is responsible for this disease.3-5

SAP, a small cytoplasmic protein that contains a single SH2-domain, is expressed at high levels in

T and NK cells. SAP interacts through its SH2 domain with tyrosine residues located in the

cytoplasmic domains of several molecules of the SLAM/CD2 receptor family, including SLAM,5

2B4,6 NTB-A,7 CD84 and Ly9,8,9 and CRACC.10 Earlier studies showed that SAP regulates the

signal transduction of the SLAM/CD2-family receptors by interfering with the recruitment of the

tyrosine phosphatase SHP-2.5,11 Recently, compelling evidence was provided that SAP behaves as

a signalling adapter permitting the recruitment of the protein tyrosine kinase FynT11-14 and the 5'

inositol phosphatase SHIP.12,15 In XLP patients, the SAP protein is either absent or structurally

altered due to germline mutations of the SAP gene, and may thus fail to regulate the above-

mentioned signalling events.3-5,16-19

sap-deficient mice have been generated to study the cellular basis of XLP. In two sap-deficient

mouse models, infection with lymphocytic choriomeningitis virus (LCMV) or T. gondii led to

hyper-proliferation of IFN-γ-producing T cells and decreased antibody responses.20-22 We have

reported the generation and characterization of a third sap-deficient mouse model that displays

hyper-activation of the CD8+ cells and decreased level of total serum IgE and IgG both before and

after murine gammaherpesvirus 68 (MHV-68)-infection.23 Moreover, a defect in NKT development

has been observed in the above mouse models and in humans.24-26 Previous investigations revealed

an altered in vivo Th2 cytokine profile in sap-deficient mice, indicating that impaired Th2

differentiation accounts for the uncontrolled expansion of Th1 cells20-23, as well as the impaired Ig

production by B cells20,21,27 and the lack of long-lived plasma cells/memory B cells in sap-deficient

mice.22 Only recently, the widely accepted hypothesis that XLP is exclusively a T and NK defect

was challenged by Morra et al., who demonstrated defective B cell responses in the absence of SAP

by an in vivo approach.28 In parallel, we have addressed the question of whether SAP has a direct

role in B cells by an in vitro assay. In the present study, we report the impaired Ig switch

recombination in sap-deficient B cells, indicating an intrinsic B cell defect.




4

Materials and Methods

Mice

The generation of the sap-deficient mice in 129/Sv X C57BL/6 mixed background was previously

reported.23 The mutant mice were back-crossed to a C57BL/6 background for more than 10

generations. Mice were bred and housed under specific pathogen-free conditions at the IARC. Age-

matched mutant and wild-type male mice, aged two to six months, were used.

Flow cytometry

Single cell suspensions from indicated organs were depleted of erythrocytes by lysis in hemolytic

gey’s solution. 1.0 x 106 cells were pre-incubated with Fc block (rat IgG2b anti-CD16/32, clone

2.4G2, BD/PharMingen, San Diego, CA) for 5 min at 4°C prior to staining with antibodies

conjugated to fluorescein isotyiocyanate (FITC), phycoerythrin (PE), tricolor (TC), allophycocyanin

(APC), or biotin. Biotin conjugates were visualized with fluochrome conjugated-Strepavidin

(BD/PharMingen). The following anti-mouse mAbs were purchased from BD/PharMingen, Caltag

(Burlingame, CA), or SouthernBiotech (Birmingham, AL): anti-B220 (RA3-6B2), anti-CD43 (S7),

anti-BP-1 (6C3), anti-CD24 (M1/69), anti-IgM (R6-60.2), anti-IgD (11-26c.2a), anti-CD5 (53-7.3)

anti-CD21/CD35 (7G6), anti-CD23 (B4B4), anti-CD69 (H1.2F3), anti-CD3e (500-A2), anti-CD4

(CT-CD4), anti-CD8a (CT-CD8a), anti-NK1.1 (PK136), anti-2B4. Ten thousand cells were

acquired on a FACSCalibre flow cytometer (BD Labware, Franklin Lakes, NJ) and analyzed with

WinMDI.

Positive selection of B cells and amplification of the sap transcript

Splenocytes were stained with anti-CD3-FITC (500-A2) and anti-CD19-Tricolor (6D5) (Caltag),

and sorted into CD19+ and CD3+ populations using FACSVantage SE (BD). Total RNA was

prepared from the sorted cells by RNeasy kit (QIAGEN, Hilden, Germany) and was reversed

transcribed by GeneAmp RT-PCR Core kit (Applied Biosystems/Roche, Branchburg, NJ). The

SAP,23 β–actin,29 and CD330 transcripts were amplified as described previously.

Negative selection of B cells and Western blot hybridization

Splenocytes were stained with PE-conjugated anti-mouse CD3e (500-A2), CD4 (GK1.5), CD8a

(53-6.7), NK1.1 (PK136), and 2B4 (BD/PharMingen). The cells positive for the above markers

were depleted by the MACS anti-PE MicroBeads and MACS separation columns (Milteni Biotec,

Bergisch Gladbach, Germany). After the depletion, plastic-adhering cells were removed by




5

incubation at 37oC for 1.5 hr. SAP protein in cell extracts after immunoprecipitation was visualised

by immunoblotting using polyclonal rabbit anti-SAP antibodies as described previously.12

Purification and culture of the small resting B cells

B cells were purified in two stages. First, they were negatively selected from splenocytes by

immuno-magnetic depletion with mouse Pan T, CD4, and CD8 beads from Dynal Biotech (Oslo,

Norway). Second, small resting B cells were fractionated by centrifugation through a discontinuous

Percoll gradient.31 0.5 x 106 cells/ml were stimulated in 96-well plates, 24-well plates or T75 cell

culture flasks with 10 µg/ml of LPS (Sigma, St. Louis, MO) or 1 µg/ml anti-mouse CD40

(BD/PharMingen), in combination with the following cytokines from R&D Systems (Menneapolis,

MN): 10 ng/ml of IL-4, 10 ng/ml of IFN-γ, or 0.5 ng/ml of transforming growth factor-β1 (TGF-

β1). To obtain an optimal cell growth, stimulation by TGF-β1 is carried out in the presence of 8

ng/ml of IL-4 and 0.25 ng/ml of IL-5, as described by Snapper et al.32 The cells were cultured in

RPMI 1640 medium or DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM

glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptolethanol, 1 mM sodium pyruvate,

100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen/GIBCO, Groningen, The

Netherlands). The proliferative response of B cells under the above conditions was measured by

assessing [3H] thymidine incorporation.

Measurement of immunoglobulin secretion and circular transcripts

Immunoglobulin (Ig) titers from the supernatant of cultured B cells (1.0 x 106 cells/24 well at the

start of stimulation) were determined by ELISA as previously described,23 except for IgG2a, whose

counter part is IgG2c in C57BL/6 mouse background.33 For measuring IgG2c, goat anti-mouse IgG

(H+L) (Jackson Immunoresearch Laboratories, West Grove, PA) was used as capture antibody, and

HRP-conjugated goat anti-mouse IgG2c (SouthernBiotech) as detecting antibody. 20 x 106 cells

were harvested at different time points after stimulation. Total RNA was extracted from the

stimulated cells by TrizolTM Reagent (Invitrogen), and was reversed transcribed as described above.

The circular transcripts of Iα and all subtypes of Iγ were amplified following the protocols reported

by Kinoshita et al.34 The β–actin transcript was amplified by PCR conditions described

previously.29




6

Results

Normal B cell development in sap-deficient mice

The striking defect in the three sap-deficient mouse models is the dysregulation in Ig production.20-

23 We previously showed comparable cell numbers and percentages of CD19+ as well as CD3+,

CD4+, and CD8+ populations in sap-deficient and wild-type littermate mice.23 In order to study

whether impaired Ig production is due to a lack of specific B cell subtypes, in addition to defective

CD4+ function, we analyzed the B cell subsets in the bone marrow, spleen, and peritoneal cavity. As

shown in Fig. 1, the proportions of early B cells in the bone marrow (using Hardy fraction

classification:35 B220/CD43/BP-1/CD24) (Fig. 1a), mature B cells in the spleen (B220+IgM+,

IgM+IgD+, IgM+CD5+, IgM+CD21+, and CD21+CD23+ cells) (Fig. 1b), and peritoneal B1

population (B220+CD5+) (Fig. 1c) were comparable in wild-type and mutant mice, indicating that

the major B cell sub-types develop normally in sap-deficient mice.

Expression of SAP in B cells

To address whether SAP plays a role in B cell function, we assessed SAP expression in wild-type B

cells. We purified B lymphocytes and performed RT-PCR and immunoprecipitation/immunoblot to

examine sap mRNA and SAP protein expressions. Using positive selection by FACS sorting, we

obtained a CD19+ population with less than 0.12% of the CD3+, CD4+, CD8+, NK1.1+, and 2B4+

cells (data not shown). Using negative selection, we obtained B cell preparations with less than 1%

of the above contaminants (data not shown). Semi-quantitative RT-PCR analysis of RNA prepared

from the sorted CD19+ cells and from the negatively enriched B cells revealed the presence of the

sap mRNA, albeit at a lower level than in thymocytes, splenocytes, or sorted CD3+ cells (Fig. 2a).

Interestingly, sap expression was down-regulated after B cell stimulation with LPS plus IL-4 (Fig.

2a). Due to the limited number of cells available from FACS-sorting, the SAP protein was studied

in negatively selected B cells. Consistent with the aforementioned expression of the sap mRNA

(Fig. 2a), the SAP protein was present at low levels in B cell extracts, and decreased gradually

following LPS plus IL-4 stimulation (Fig. 2b). To test the purity of the enriched B cells, CD3+

transcript was measured in the RNA samples by RT-PCR. While no amplification band was found

in the positively selected B cells or in the B cells after stimulation, a very faint amplification band

was detected in the negatively enriched B cell preparation (Fig. 2a). Subsequently, we performed a

mixing experiment to rule out the possibility that the sap-transcript or the SAP protein observed in

the above assays originate from trace T cell contamination. Purified B cells from sap-deficient mice

that are devoid of SAP were mixed with T cells from wild-type mice to mimic various extents of T

cell contamination and then examined for SAP protein expression. As shown in Fig. 2b, 1% and 2%




7

of T cell contamination generate faint signals in comparison to SAP expression in wild-type B cell

preparations. Only when T cell contamination reaches 4% does T cell-derived SAP signal become

comparable with that from the wild-type B cells. Since, as shown by FACS analysis, T cells

represent less than 1% of the enriched B cell population used in our experiments, it could be

excluded as the source of the positive SAP signal in purified B cells.

Decreased IgG and IgA production in sap-deficient B cells

Since SAP was shown to be expressed in wild-type B cells, we investigated the impact of SAP-

deficiency on B cell function, in particular, Ig production. In order to eliminate any in vivo pre-

programming/interference, we used small resting B cells, which represent the mature naïve B cell

population. The small resting B cells were isolated from the spleen by negative selection followed

by density centrifugation. The resulting cell population was 92% B220+ and contained less than

0.8% CD3+ cells (Fig. 3), as assessed by flow cytometry. Of the B220+ cells, 98% were IgM+, and

94% were IgM+IgD+ (Fig. 3). The percentages of the B220+CD43+ and B220+CD69+ populations,

representing the mature activated B cells, were low in both sap-deficient and wild-type cell

preparations (Fig. 3). In order to study Ig production, these isolated small resting B cells were

stimulated for 6 days with LPS in combination with IL-4, IFN-γ, or TGF-β1, and the secreted

immunoglobulins were measured by ELISA. As shown in Fig. 4, the IgM and IgE levels were

comparable in both genotypes; however, IgG and IgA production was impaired in sap-deficient B

cells under most conditions. In particular, the levels of secreted IgG2c (equivalent to IgG2a in

C57BL/6 background33), IgG2b, IgG3, and IgA were significantly lower in sap-deficient B cells

compared to wild-type B cells (Fig. 4).

In order to rule out the possibility that the decreased in vitro Ig production was due to poor

outgrowth of the sap-deficient B cells during culture, we assessed the proliferation rates of the

cultured cells by measuring the incorporated [3H]-thymidine. As shown in Fig. 5, the proliferation

rates of sap-deficient and wild-type B cells were comparable. Moreover, numbers of sap-deficient

and wild-type live B cells, determined by trypan blue exclusion, were similar at various time points

(data not shown).

Impaired Ig class switch recombination in sap-deficient B cells

Subsequently, we addressed whether the decreased IgG2c, IgG2b, IgG3, and IgA production is due

to a defect of Ig class switch recombination (CSR). Kinoshita et al. demonstrated that CSR can be

determined by measuring the abundance of RNA transcripts from the looped-out circular DNA.34

We studied the expression level of Iγ2a, Iγ2b, Iγ3, and Iα circular transcripts in sap-deficient and




8

wild-type B cells activated by LPS in combination with IL-4, IFN-γ, or TGF-β1. All four

stimulation conditions led to detectable levels of Iγ2b and Iγ3 transcripts after 36 hours of treatment;

however, transcript levels were lower in sap-deficient B cells than in wild-type B cells (Fig. 6a).

Similarly, Iγ2a and Iα transcripts, which are clearly induced after three days of stimulation by IFN-γ

and TGF-β1, respectively, were found significantly lower in sap-deficient B cells than in wild-type

B cells (Fig. 6a). In addition, we analyzed the response of B cells following a more physiological

stimulation, anti-CD40 in combination with the three cytokines. All the aforementioned circular

transcripts were measured. Iγ2b and Iγ3 were undetectable (not shown). However, Iγ2a and Iα

transcripts were dramatically lower in anti-CD40+IFN-γ and anti-CD40+TGF-β1 stimulated sap-

deficient B cells as compared to wild-type B cells (Fig. 6b).




9

Discussion

To study the role of SAP in the immune system, several laboratories including ours have generated

sap knockout mice.20,21,23 Although there are differences in the gene targeting strategies, all three

sap-deficient mice have basal Ig deficiencies. Previous investigations20,21 attributed the Ig

disregulation in sap-deficient mice to a defect in Th2 function. Following these studies, Cotty et al.

demonstrated that defective CD4+ T cell help is responsible for the lack of long-lived plasma cells

and memory B cells in sap-deficient mice,22 Hron et al. showed that only the T-dependent antibody

responses are impaired in sap-deficient mice,27 and more recently, Ma et al. reported the lack of

isotype-switched memory B cells in the peripheral blood of the XLP patients, which is associated

with defective IL-10 production by CD4+ T cells.36 However, whereas the T cell defect has been

unequivocally demonstrated, none of these studies have excluded the possibility of an intrinsic

functional defect in a subset of sap-deficient B cells. To address this question, we examined the B

cell compartment in sap-deficient mice. Purified small resting B cells were used to study in vitro Ig

production following stimulation with LPS and anti-CD40 in combination with IL-4, IFN-γ and

TGF-β1. We observed impaired IgG and IgA production in sap-deficient B cells, and we

demonstrated that this was not a result of either abnormal growth during the cell culture or altered

development of the major B cell sub-populations. Thus these data expand the role of SAP in

regulating cellular functions to include the B cell compartment. Consistent with our finding, a

recent study provides convincing evidence demonstrating the presence of defective B cell responses

in a sap-deficient background by adoptive transfer of wild-type and sap-deficient T and B cells.28

While some studies reported the absence of the SAP protein in B cell lysates,37,38 others showed that

SAP is expressed in B cells of the inter-follicular zone of the tonsil and lymph nodes of the

humans39 and in follicular GC B cells in the spleen and lymph nodes in humans4 or in the mouse.28

Moreover, it has been shown that the SAP protein is detectable in Hodgkin and non-Hodgkin

lymphoma, tonsillar B cells, some B cell lines, and Burkitt lymphoma cell lines.4,15,38,39 We

observed the expression of the sap mRNA and the SAP protein in B cells from wild-type mice,

although at very low levels (Fig. 2). We also showed that activation of wild-type, mature naïve B

cells by LPS in the presence of IL-4 down-regulates the transcript and the protein product of the sap

gene. This result indicates that SAP is a tightly controlled molecule, expressed transiently in

specific stage(s) of B cell differentiation. The exact subset of B cells expressing SAP and the

signaling pathways that are regulated by SAP in B cells remain to be elucidated. One plausible

model is that SAP regulates B cell function by interacting with SLAM, as engagement of SLAM in

B cells has been shown to enhance B-cell proliferation and Ig production.40 This is supported by the

result of Mikhalap et al., who showed that SAP and SLAM are co-expressed in B cells in the




10

interfollicular zone.39 It is also possible that SAP interferes with B cell function through CD84

signaling, which is up-regulated in memory B cells and recruits SAP.8,9 Finally, all the SLAM

family members with the exception of 2B4 are expressed on B cells, and are therefore good

candidates for further investigation.

It is well established that LPS stimulation of B cells in the presence of cytokines can induce in vitro

isotype switching.41 Results in Fig. 4 demonstrate that sap-deficient B cells produce lower levels of

IgG2c, IgG2b, IgG3, and IgA in vitro than do wild-type B cells. This defect is not caused by

abnormal proliferation of sap-deficient B cells in response to the stimulation (Fig. 5); instead, it

reflects an impaired function of B cells. To exclude the possibility that the reduction in Ig

production of sap-deficient B cells was a result of a decrease in the number of memory B cells, we

used small resting B cells, which were principally B220+IgM+ (98%). The proportion of B220+IgM-

cells, which might represent the switched IgG+ or IgA+ cells, was very low and comparable in sap-

deficient and in wild-type B cell preparations. We also excluded the possibility that decreased Ig

production in sap-deficient B cells is due to a lower percentage of activated B cells before

stimulation, since percentages of the B220+CD43+ and B220+CD69+ populations are equally low in

sap-deficient and in wild-type B cells (Fig. 3). It is worth noting that the impairment of Ig

production in sap-deficient B cells is only partial. Consistent with our observations, Morra et al.

observed that while the follicular GC B cells are affected in sap-deficient mice, mature B cells

outside the GC and T-independent responses are normal.28 Therefore, one could speculate that the

absence of SAP leads to a defect in Ig class switch only in the subset of B cells expressing SAP and

that this defect is partially masked by the presence of functionally unaltered B cells in the total B

cell compartment.

To study the mechanism of the impaired Ig secretion, we measured the CSR rates in sap-deficient

and wild-type B cells to study the isotype switching process. After cytokine stimulation, CSR is

accompanied by looping-out DNA segments between the Cµ switch region and one of the

downstream switch regions of each CH gene, including its I promoter.34 Kinoshita et al. have shown

that the I promoter is active in the excised circular DNAs and directs the production of circular

transcripts, which are sensitive molecular markers of active CSR.34 We observed decreased levels of

Iγ2a, Iγ2b, Iγ3, and Iα circular transcripts in sap-deficient B cells stimulated with either LPS or anti-

CD40 in combination with IL-4, IFN-γ, or TGF-β1. This result indicates a defect in Ig class switch

in sap-deficient B cells, which is not Th1- or Th2-cytokine dependent. We further tested other

components of Ig transcriptional machinery, e.g. AID and T-bet, in sap-deficient mice. The

activation-induced cytidine deaminase (AID) gene is expressed in activated B cells and essential to




11

both CSR and somatic hypermutation (SHM).42-44 The T-box transcription factor T-bet is involved

in IgG class switch, especially IgG2a, by regulating germ-line transcription.45,46 Interestingly, no

differences in AID and T-bet expression were observed between wild-type and sap-deficient B cells

(data not shown). Thus, other pathway(s), which remains to be determined, may be responsible for

the impaired Ig class switch in B cells from sap-deficient mice. In exploring the potential role for

SAP in CSR, one must consider the finding of Sylla et al., who demonstrated that the wild-type

form of the SAP protein has an affinity for Ku70 and Ku80 proteins,47 which are known to be

required for CSR.48,49 Therefore, one possibility is that SAP may be linked to some components of

the CSR machinery.

Taken together, the impaired in vitro Ig production in sap-deficient B cells and the decreased level

of CSR suggest a B cell dysfunction in sap-deficient mice in addition to the abnormal CD4+ T cell

help previously reported.20-22,27 Its presence in wild-type B cells further supports the concept that the

adaptor molecule SAP directly regulates B cell function. This significant finding may provide novel

insights into the pathology and/or mechanism of the XLP disease.




12

Acknowledgements

This work is dedicated to our late colleague Jun Liang. We thank Dr Giovanni Romeo (IARC,

Lyon) for his encouragement and support, Mr Jean-Jacques Medard (IARC) for technical assistance,

Miss Lily Wang (IARC) and Dr Benoit Pasquier (Unité INSERM 429, Paris) for some of their

work, and Mrs Antoinette Trochard (IARC) for assistance in preparing the manuscript. We are

grateful to Drs Uzma Hasan, Eric Garcia (Schering-Plough, Dardilly, France), Mrs Francoise Le

Deist (INSERM 429) for help with FACS-sorting, and Drs Anne Durandy (INSERM 429), Bao

Vuong, and Paul Rothman (Columbia University, New York) for stimulating discussions and

critical reading of the manuscript.




13

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Legends

Figure 1. B cell sub-populations in the bone marrow (a), spleen (b), and peritoneal cavity (c).

a, Percentages of gated cells or percentages of each fraction by Hardy classification35 (A, B, C, and

C’) are indicated. b and c, Percentages of different B cell sub-populations are indicated in the

quadrants.

Figure 2. Expression of SAP in B cells. a, Expression of the sap transcript. RT-PCR products with

primers 91U and 457L23 from 1µg of total RNA extracted from the following cells: thymocytes (T),

splenocytes (S), sorted CD3+ cells (CD3), sorted CD19+ cells (B1), negatively selected B cells,

unstimulated (B2), and stimulated by LPS+IL-4 for 3 days (D3) and 6 days (D6) respectively. Mouse

fibroblast cell line NIH 3T12 (F) and mouse neuroblastoma cell line Neuro 2A (N) were used as

negative controls for sap expression. The transcript of the mouse β–actin gene was used as the

amplification control, and the transcript of mouse CD3 as the purity control of the enriched B cells. b,

Expression of the SAP protein. Upper part, SAP protein detected from thymocytes (T), splenocytes

(S), T cells selected by Dynal beads (Tcell), negatively selected B cells, unstimulated (B), and

stimulated with LPS+IL-4 for 6 hr (6h), 18 hr (18h), 3 days (D3) and 6 days (D6), respectively; lower

part, SAP protein detected from negatively selected wild-type B cells (B cells SAP+) and from

negatively selected sap-deficient B cells (B cells SAP-) mixed with wild-type T cells mimicking

various percentages of T cells contamination. The same amount of cell extract (0.5 mg protein) was

immunoprecipited by anti-SAP antibody and the Western blot was hybridized with the same anti-SAP

antibody

Figure 3. Purity of the isolated small resting B cell preparations. Left column, wild-type B cells

(sap+); right column, sap-deficient B cells (sap-). Percentage of cells positive for selected surface

markers is indicated in each quadrant.

Figure 4. Aberrant Ig production by sap-deficient B cells after in vitro activation. Small resting

B cells from wild-type (open circles) and from sap-deficient mice (filled circles) were stimulated for

6 days with LPS plus different cytokines as indicated. Each circle represents the concentration

(µg/ml) of secreted Ig in the supernatant of the cells stimulated by the given culture condition. The

average value of each isotype is indicated by a horizontal bar. Student T test *P<0.05.

Figure 5. Proliferation rates of the stimulated wild-type and sap-deficient B cells. Open and

filled columns represent the wild-type and sap-deficient B cells, respectively. Cells were cultured




19

for 3 days or 6 days. Average values (CPM) of incorporated [3H]-thymidine, ± standard deviations,

are shown. A similar pattern was observed in three other independent experiments.

Figure 6. Circular transcripts generated by activated B cells. (a) Circular transcripts detected

after LPS plus cytokine stimulation. Iγ2b, Iγ3 were analyzed after 36 hrs’ stimulation; Iγ2a and Iα

after 3 days. (b) Iγ2a and Iα production after 3 days of stimulation with anti-CD40 plus cytokine.

RT-PCR products were amplified from 1µg of total RNA extracted from stimulated cells. The same

patterns were observed in at least three independent experiments. LB, large activated B cells

isolated by centrifugation on the Percoll gradient; SB, small resting B cells purified by Percoll

gradient; the remaining samples were from small resting B cells stimulated in vitro. W, wild-type B

cells; M, sap-deficient B cells. The transcript of the mouse β–actin gene was used as the

amplification control.




/-

92.2

0.63

0.43 92.1

0.42

0.84

78.1

0.02

14.7 77.4

0.25

15.2

0.26

0.28

91.8 0.43

0.12

92.7

66.6

0.11

26.3 68.7

0.07

24.4

3

0

% of total cells in bone marrow

sap+ sap-

4

BP-1 C C’

BA

30.09.6 10.9

44.9

% of gated B220+/CD43+ cells

8.57.930.248.8

Fig. 1

(a)

(b)

(c)

sap+ sap-

sap+ sap-

3.6

CD

CD21/35-FITC

7.2 8.6

sap+

sap-

49.5 24.0

16.7

23.956.0

11.6


CD2135FITC

CD21/35-FITC

23-

CD4

B22

CD23-

4.9

42.6

CD21/35-FITC

45.6

CD2

20



21

(a) LPS+IL-4

T S CD3 B1 B2 D3 D6 F N

LPS + IL-4T S Tcell B 6h 18h D3 D6

(b)

SAP

sap

β-actin

Fig. 2

B cells B cells SAP+ T cells SAP+ SAP- 1% 2% 4%

CD3

SAP




22

sap+ sap-

90.0

0.71

1.9 90.7

0.50

2.0

4.1

1.6

86.6 87.73.6

1.3

1.6

91.5

0.04 1.7

93.4

0.10

2.4

90.7

5.4 2.13.9

92.9

0.19

0.19

0.57 0.16

0.19

0.16

Fig. 3




23

IgG1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

IgG2c

0

0.5

1

1.5

2

2.5

IgG2

0

0.5

1

1.5

2

2.5

3

IgG3

0

0.1

0.2

0.3

0.4

0.5

0.6

IgM

0

0.5

1

1.5

2

2.5

3

3.5

4

IgE

0

1

2

3

4

5

6

7

8

9

IgA

0

0.2

0.

0.7

1

LPS+IL-4Med LPS LPS+IFN-γ LPS+TGF-β1

Med LPS LPS+IFN-γLPS+IL-4

Med LPS LPS+IFN-γLPS+IL-4

*

*

*

*

*

*

Fig. 4




24

Fig. 5

0

5000

1000

1500

sap

day 3

0

1000

2000

3000

4000

5000

day 6

Medium - +IL-4 +IFN +TGFLPS

sap-CP




Unstimulated LPS LB SB - +IL-4 +IFN-γ +TGF-β1W M W M W M W M W M W M

Fig. 6

a(a)

a

αCD40 - +IL-4 +IFN-γ +TGF-β1

W M W M W M W M

(b)


Iγ2

α
I
Iγ3

Iγ2b

n
β-acti
Iγ2

Iα

β-actin

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Impaired Ig class switch in mice deficient for the X-linked lymphoproliferative disease gene Sap

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