Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract Full Text (PDF) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JEM Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ferrari, S. Articles by Plebani, A. Search for Related Content PubMed PubMed Citation Articles by Ferrari, S. Articles by Plebani, A. Social Bookmarking                      What's this?

¹ Medical Genetics Unit, S. Orsola-Malpighi University Hospital, 40138 Bologna, Italy
² Department of Pediatrics and ³ Institute of Molecular Medicine A. Nocivelli, University of Brescia, 25123, Brescia, Italy
⁴ Department of Molecular Immunology, Faculty of Biology, and ⁵ Max-Planck-Institute for Immunobiology, Albert-Ludwigs-University Freiburg, 79108 Freiburg, Germany
⁶ Pediatric Pneumology and ⁷ Cystic Fibrosis Center, Ospedale Civile Maggiore, 37126 Verona, Italy

CORRESPONDENCE Simona Ferrari:simona.ferrari{at}med.unibo.it;OR Alessandro Plebani:plebani{at}med.unibs.it

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Agammaglobulinemia is a rare primary immunodeficiency characterized by an early block of B cell development in the bone marrow, resulting in the absence of peripheral B cells and low/absent immunoglobulin serum levels. So far, mutations in Btk, µ heavy chain, surrogate light chain, Ig, and B cell linker have been found in 85–90% of patients with agammaglobulinemia. We report on the first patient with agammaglobulinemia caused by a homozygous nonsense mutation in Igß, which is a transmembrane protein that associates with Ig as part of the preBCR complex. Transfection experiments using Drosophila melanogaster S2 Schneider cells showed that the mutant Igß is no longer able to associate with Ig, and that assembly of the BCR complex on the cell surface is abrogated. The essential role of Igß for human B cell development was further demonstrated by immunofluorescence analysis of the patient's bone marrow, which showed a complete block of B cell development at the pro-B to preB transition. These results indicate that mutations in Igß can cause agammaglobulinemia in man.

The development of B lymphocytes from pluripotent progenitors is a tightly regulated process that occurs in hemopoietic tissues, primarily embryonic liver and bone marrow in mammals (1). In these sites, lymphoid progenitors lacking Ig expression (pro-B cells) give rise to large B lymphocyte precursors (preB cells) expressing µ heavy chains (µHCs) (2–5) as a result of µHC V(D)J gene rearrangements. A key checkpoint in B cell lineage development is the ability of the newly generated µHC to associate with the surrogate light chain (SLC) composed of VpreB and 5/14.1 and homologous to the V and C regions of LCs (6, 7). SLC binds nascent µHC proteins, thereby releasing them from BiP-mediated retention in the endoplasmic reticulum (8–9). SLC/µHC homodimers then associate with Ig/Igß heterodimeric signal-transducing elements to form the preB cell receptor (BCR), which is exported from the Golgi apparatus to the preB cell surface in the context of lipid rafts, where they associate with signaling elements such as Syk, Lyn, Btk, and B cell linker (BLNK). Signaling through the preBCR leads to a transient cellular proliferation and the V-J_L rearrangement of the or LCs (10–12). Successful VJ_L rearrangement allows the assembly of BCRs composed of µHC, LC, and Ig/Igß on newly generated immature IgM-expressing B cells (13–14), which then exit the bone marrow and complete their maturation in the secondary lymphoid organs.

The crucial role of preBCR signaling during early B lineage differentiation is indicated by the block in pro-B to preB cell differentiation in mice and man with deficiencies in single preBCR components or in the essential downstream signaling elements (15–20).

Mutations of BTK, which is the gene responsible for X-linked agammaglobulinemia, account for 85% of cases with agammaglobulinemia and absent circulating B cells (21). The remaining 15% of patients constitute a heterogeneous group, including patients with mutations in µHC, 5, Ig, and BLNK, which are the genes responsible for the autosomal recessive forms of agammaglobulinemia. However, mutations in these genes do not account for all patients with selective defect of B cell development, suggesting that mutations in other, yet to be identified genes may affect early B cell development. Regardless of the genetic defect, patients with a selective arrest of B cell development are characterized by early onset of severe bacterial infections as a result of their inability to mount an antibody response.

To better clarify the nature of genetic defects that results in abnormal B cell development in man, we screened patients with early-onset hypogammaglobulinemia and absent circulating B cells for mutations in genes that are expressed in the early stages of B cell differentiation.

We have identified one patient with a homozygous defect in the Igß-encoding gene, and have compared the peripheral B cell phenotype of this patient with that of patients with defects in the µHC or Btk. Transfection experiments using D. melanogaster S2 Schneider cells demonstrated that the mutation in Igß abrogates the assembly of the BCR on the cell surface. Bone marrow studies showed that the mutation causes a complete block in B cell development at the pro-B to preB transition, a phenotype resembling that observed in Igß-null mice (22).

	RESULTS AND DISCUSSION

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mutation of the Igß gene causes agammaglobulinemia
As part of an ongoing effort to extensively genotype agammaglobulinemic patients for known and candidate genes causing agammaglobulinemia (23), we found for the first time a homozygous mutation in the Igß-encoding gene in a patient clinically diagnosed with agammaglobulinemia. The patient, a 20-yr-old Italian male, is the first-born child of healthy parents without known consanguinity. He has two healthy sisters, and no positive family history for primary immunodeficiencies is present in the pedigree. The pregnancy and delivery were uneventful. At the age of 8 mo, the patient was hospitalized because of pneumonia of the left lobe and Salmonella-caused enteritis. Immunological work up showed a marked hypogammaglobulinemia (IgG, 100 mg/d; IgA and IgM, undetectable) and absence of peripheral CD19-positive cells (<1%), in the presence of normal numbers of T and NK cells; T cell function, assessed by mitogen-induced proliferation, was normal. Intravenous immunoglobulin replacement therapy was initiated at the age of 8 mo. During follow up, despite appropriate immunoglobulin substitution therapy, the patient suffered several episodes of bacterial conjunctivitis, acute otitis media, sinusitis, and bronchitis. A computed tomography scan of the nasal sinuses showed a pattern compatible with chronic sinusitis, whereas no bronchiectasis was observed. BTK gene sequence analysis was normal.

Genomic DNA from this patient was analyzed by PCR amplification and direct sequencing of exons and exon–intron junctions of genes encoding for the preBCR components. We found a homozygous C>T nucleotide substitution in exon 3 of the B29 gene, encoding for the Igß protein (Fig. 1), at position c.238 (GenBank accession no. M89957) corresponding to amino acid 80. The mutation causes the replacement of Gln80 with a stop codon within the extracellular immunoglobulin domain of Igß, preventing the expression of the functional transmembrane protein and possibly interfering with the assembly of the preBCR on cell surface.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Mutation analysis of the B29 gene. (A) Electropherograms from a portion of exon 3 showing the 238 C>T mutation at the base corresponding to amino acid 80 of the Igß chain, compared with the wild-type sequence from a healthy donor (WT). The sequences obtained from heterozygous parents (I.1 and I.2) are shown. (B) Schematic representation of Igß-encoding gene.

 
Both parents of the patient were heterozygous for this mutation. The Gln80X mutation was not detected in DNA samples obtained from 90 healthy controls; in addition, sequence analysis of the whole B29 gene reveals the presence of a common haplotype represented by a T>C silent change at codon 122 in cis with a 3'-untranslated region T>C transition, which is present at a high frequency (f_CC = 0.76) in the 90 controls. Our patient is homozygous for the less common 122/T and 3'-untranslated region /T haplotype, suggesting the possibility of a founder effect for the mutated allele. Although not reported, consanguinity of patient's parents cannot be excluded.

Reconstitution of the BCR in S2 cells
It has been shown that efficient transport of IgM to the cell surface requires coexpression of Igß furthermore, IgM and Igß alone are sufficient to reconstitute antigen-specific signal transduction by Igß (24). The effect of the Gln80X Igß mutation on BCR assembly was assessed by a reconstitution experiment in Drosophila melanogaster S2 cells.

The human Igß cDNA was cloned in an appropriate expression vector, and D. melanogaster S2 cells were cotransfected with mouse 1LC, µHC, FLAG-tagged Ig, and either human wild-type Igß or mutant Gln80X Igß. After induction of expression of the recombinant proteins, cells were harvested and reconstitution of the IgM BCR was detected by staining with a phycoerythrin (PE)-conjugated anti-FLAG antibody that binds to Ig and a biotinylated anti-IgM antibody. PE- and Cy5-conjugated streptavidin double-positive cells indicate the expression of the BCR on the cell surface.

Fig. 2 shows that the wild-type human Igß chain is as efficient as the mouse Igß chain at promoting assembly of the IgM BCR complex on the cell membrane (compare the bottom left and top right graphs). On the contrary, insertion of the Gln80X mutation in Igß prevents reconstitution of the IgM BCR on the cell surface, which is similar to expression of Ig only (compare the bottom right and top left graphs).

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Surface expression of the IgM BCR in S2 cells. S2 cells were cotransfected with plasmids encoding for EGFP, mouse µHC, 1 LC, Ig, and different Igß chains, as indicated. After 5 h, expression cells were harvested and stained with PE-labeled anti-FLAG antibody and biotinylated anti-IgM antibody, followed by streptavdin-Cy5, to detect Ig and membrane IgM, respectively. Cells were gated for EGFP-positive cells to normalize the transfection efficiency.

 
S2 cells cotransfected with FLAG-tagged Ig and wild-type Igß and stained with the PE-conjugated anti-FLAG antibody showed that the human Igß alone was not able to carry Ig to the cell surface, proving that, in the aforementioned experiments, any Ig chain detected at the S2 cell surface is, indeed, part of the assembled BCR complex (unpublished data).

Immunofluorescence studies
PBMCs from the patient carrying the Igß mutation, three patients with Btk deficiency, three patients with µHC deficiency, and three healthy controls were compared using standard flow cytometry markers. The dramatic reduction of circulating B cells in the Igß-deficient patient was confirmed using the typical B cell marker CD19 (unpublished data). As expected, PBMCs from the patient, as well as PBMCs from patients with mutations in Btk and µHC, did not express mature IgM on the cell surface (unpublished data).

The analysis of the alternative pan–B cell marker CD22 in the Igß-deficient patient, as well as in Btk- and µHC-deficient patients, revealed the presence of a cell population expressing CD22 at lower levels compared with healthy controls (unpublished data). The CD22^lo subset was poorly represented (<1%) in 25 healthy controls, whereas in patients CD22^lo cells range from 2 to 4.5% of the gated lymphocytes.

The CD22^lo cells did not express either the pan–B cell marker CD19 or the early B cell marker CD10; because the CD22 molecule was previously found expressed in human basophiles (25), we tested if the CD19–/CD22^lo population in agammaglobulinemic patients was accounted for by basophiles. This turned out to be the case, as demonstrated by Toluidine blue staining of the sorted CD22^lo cells, showing pure basophiles with typical large, dark granules. Furthermore, the CD22^lo cells were positive for CD13, which is a myelomonocytic lineage marker (unpublished data). Therefore, the CD22^lo cell population from the Igß-deficient patient did not belong to the B lineage.

To determine at which stage B cell development is blocked, bone marrow cells from the patient and an age-matched control were evaluated by cytofluorimetric analysis. CD19+ B cells were markedly reduced in the patient (1%) when compared with the healthy control (>10%) (Fig. 3). Two-color immunofluorescence was used to define the percentages of pro-B cells (CD34+CD19–) and preB/immature B cells (CD34–CD19+). The coexpression of CD19 and CD34 showed that >85% of the patient's B cells belong to the pro-B stage of differentiation, whereas <5% of the healthy control's B cells presented the pro-B phenotype. These findings indicate a block of B cell development at the transition from the pro-B to preB stage.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Cytofluorimetric analysis of B cell development. Bone marrow mononuclear cells from the Igß-deficient patient and an age-matched healthy control were stained for surface CD19, CD34, Ig, and intracellular TdT. The patient showed a marked reduction in the number of CD19+ cells within the lymphoid gate, and >85% of the B-lineage cells were positive for CD34 (top row). Virtually none of the CD19+ cells from the Igß-deficient patient were B cells, as defined by the coexpression of CD19 and surface immunoglobulin (middle row). Permeabilized cells were stained for cytoplasmic TdT. The majority of the CD19+ cells from the patient were pro-B cell precursors, as defined by coexpression of CD19 and TdT (bottom row).

 
To further characterize the point of arrest of B cell development, expression of TdT and surface Ig were evaluated. Remarkably, the Igß-deficient patient presented an accumulation of TdT-positive cells that did express CD19. Furthermore, no surface immunoglobulin expression could be detected on the patient's B cells. These results further confirm that the arrest of B cell development in the Igß-deficient patient occur at the pro-B to preB transition; a similar block at the pro-B stage has been described in patients affected by mutations of other members of the preBCR complex, i.e., µHC deficiency (23) and Ig deficiency (19). Unfortunately, we were unable to perform further biochemical and functional analyses, such as assessment of the extent of V(D)J recombination, because of the low number of bone marrow lymphocyte precursors obtained from the patient biopsy.

In principle, the occurrence of a stop codon in the N-terminal portion of Igß could result in a truncated protein lacking the transmembrane domain because of premature termination; however, it is also possible that the occurence of a premature stop codon causes nonsense-mediated RNA decay. In any event, the mutation in Igß prevents the assembly of the IgM BCR on the cell surface, as demonstrated by the reconstitution experiment shown in Fig. 2.

The preBCR checkpoint is essential for B cell development. So far, animal models deficient for different preBCR components were shown to have an arrest at the pro-B to preB cell stage of development, although some models presented a leaky phenotype (e.g., the presence of peripheral B cells in 5-deficient mice [reference 26]). Human mutations in preBCR components further elucidated the role of the preBCR complex in B cell development, and underlined that animal models are not always concordant with the phenotype of patients carrying mutations in the same gene; the aforementioned human 5 deficiency presents a severe defect in B cells, with absence of peripheral B cells. On the other hand, human Igß deficiency closely resembles the phenotype observed in knockout mice in that these mice have no mature peripheral B cells and B cell development is blocked at the pro-B cell/preBI cell stage (22).

In conclusion, this is the first report of a patient with agammaglobulinemia caused by a homozygous Gln80X mutation in Igß, resulting in a stop codon that abrogates the expression of the preBCR complex on the cell surface. Bone marrow B cell analysis of the Igß-deficient patient indicated an arrest at the pro-B–preB transition, a phenotype resembling the one observed in other known forms of agammaglobulinemia.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Genetic analysis.
Genomic DNA was extracted from peripheral blood leukocytes using standard techniques. Exons and exon–intron junctions were amplified by PCR using FastStart Taq DNA Polymerase (Roche) under standard conditions. Purified PCR products were sequenced on both strands using a Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and were run on an ABI 3730 Genetic Analyzer (Applied Biosystems). The primers used in this study are available upon request. The M89957 GenBank entry was used as reference sequence.

For the genetic diagnosis, the informed consent was signed by the patient.

DNA constructs.
For inducible expression of human Igß in S2 cells, wild-type and mutant cDNAs were cloned into the S2 plasmid pRmHa-3 containing a copper-inducible metallothionein promoter. Total RNA extracted from PBMC was amplified by RT-PCR using the following oligonucleotides: Igß_cDNA-F 5'-GGTCGGGGACAGAGCAG-3' and Igß_cDNA-R 5'-CGACCTGGCTCTCACTCCT-3', and the corresponding PCR fragment was subcloned into the pRmHa-3 plasmid. The C>T single-nucleotide substitution was introduced into the Igß cDNA by site-directed mutagenesis.

Reconstitution of IgM-BCR in S2 cells.
D. melanogaster S2 Schneider cells were grown in Schneider's Drosophila medium (Life Technologies, Inc.) supplemented with 5–10% FCS at 27°C with atmospheric CO₂ levels. Transient transfection of S2 cells and induction of expression (5 h) was performed as previously described in Wossning et al. (27). For FACS analysis, cells were stained with anti-FLAG PE antibody and biotinylated anti-IgM antibody, followed by Cy5-conjugated streptavidin. To normalize the transfection efficiencies, S2 cells were cotransfected with the vector pDEGFP containing the enhanced GFP (EGFP) coding sequence.

Cytofluorimetric analysis.
PBMCs from fresh blood samples and bone marrow MCs from patients and healthy controls were isolated by Ficoll-Hypaque gradient centrifugation and stained with the following conjugated anti–human monoclonal antibodies: FITC-labeled anti–human CD19, PE-labeled anti–human CD22, PE-labeled anti–human CD34, PE-labeled anti–human surface Ig, and PE-labeled anti–human TdT. Cells were resuspended in PBS and 0.1% BSA at the concentration of 5 x 10⁵–1 x 10⁶ cells/ml, and then stained with and the anti–human antibodies using standard protocols. Stained cells were washed twice with PBS/BSA and analyzed with a FACSCalibur flow cytometer (Becton Dickinson).

The human experiments have been approved by the hospital Ethical Committee of Spedali Civili of Brescia.

	Acknowledgments

 
We acknowledge the patient and his family for their cooperation and the members of the Italian Primary Immunodeficiencies Network.

This work was supported by a grant from Fondazione Telethon to S. Ferrari. We thank the Fondazione Golgi, the Associazione Immunodeficienze Primitive and the Centro di Studio Immunodeficienze Primitive Mazio Di Martino from Brescia for their support.

The authors declare there are no conflicts of interest.

Submitted: 5 February 2007
Accepted: 24 July 2007

S. Ferrari and V. Lougaris contributed equally to this paper.

	REFERENCES

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Cooper, M.D., R.D. Peterson, and R.A. Good. 1965. Delineation of the thymic and bursal lymphoid systems in the chicken. Nature. 205:143–146.[CrossRef][Medline]
2. Owen, J.J., M.D. Cooper, and M.C. Raff. 1974. In vitro generation of B lymphocytes in mouse foetal liver, a mammalian ‘bursa equivalent’. Nature. 249:361–363.[CrossRef][Medline]
3. Osmond, D.G., and G.J. Nossal. 1974. Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling. Cell. Immunol. 13:132–145.[CrossRef][Medline]
4. Raff, M.C., M. Megson, J.J. Owen, and M.D. Cooper. 1976. Early production of intracellular IgM by B-lymphocyte precursors in mouse. Nature. 259:224–226.[CrossRef][Medline]
5. Burrows, P., M. LeJeune, and J.F. Kearney. 1979. Evidence that murine pre-B cells synthesise mu heavy chains but no light chains. Nature. 280:838–840.[CrossRef][Medline]
6. Sakaguchi, N., and F. Melchers. 1986. Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature. 324:579–582.[CrossRef][Medline]
7. Kudo, A., and F. Melchers. 1987. A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes. EMBO J. 6:2267–2272.[Medline]
8. Lassoued, K., H. Illges, K. Benlagha, and M.D. Cooper. 1996. Fate of surrogate light chains in B lineage cells. J. Exp. Med. 183:421–429.[Abstract/Free Full Text]
9. Hendershot, L.M., and J.F. Kearney. 1988. A role for human heavy chain binding protein in the developmental regulation of immunoglobin transport. Mol. Immunol. 25:585–595.[CrossRef][Medline]
10. Coffman, R.L., and I.L. Weissman. 1983. Immunoglobulin gene rearrangement during pre-B cell differentiation. J. Mol. Cell. Immunol. 1:31–41.[Medline]
11. Ehlich, A., S. Schaal, H. Gu, D. Kitamura, W. Muller, and K. Rajewsky. 1993. Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development. Cell. 72:695–704.[CrossRef][Medline]
12. Engel, H., A. Rolink, and S. Weiss. 1999. B cells are programmed to activate kappa and lambda for rearrangement at consecutive developmental stages. Eur. J. Immunol. 29:2167–2176.[CrossRef][Medline]
13. Hombach, J., T. Tsubata, L. Leclercq, H. Stappert, and M. Reth. 1990. Molecular components of the B-cell antigen receptor complex of the IgM class. Nature. 343:760–762.[CrossRef][Medline]
14. Reth, M. 1992. Antigen receptors on B lymphocytes. Annu. Rev. Immunol. 10:97–121.[CrossRef][Medline]
15. Tarakhovsky, A. 1997. Xid and Xid-like immunodeficiencies from a signaling point of view. Curr. Opin. Immunol. 9:319–323.[CrossRef][Medline]
16. Kitamura, D., J. Roes, R. Kuhn, and K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 350:423–426.[CrossRef][Medline]
17. Yel, L., Y. Minegishi, E. Coustan-Smith, R.H. Buckley, H. Trubel, L.M. Pachman, G.R. Kitchingman, D. Campana, J. Rohrer, and M.E. Conley. 1996. Mutations in the mu heavy-chain gene in patients with agammaglobulinemia. N. Engl. J. Med. 335:1486–1493.[Abstract/Free Full Text]
18. Minegishi, Y., E. Coustan-Smith, Y.H. Wang, M.D. Cooper, D. Campana, and M.E. Conley. 1998. Mutations in the human lambda5/14.1 gene result in B cell deficiency and agammaglobulinemia. J. Exp. Med. 187:71–77.[Abstract/Free Full Text]
19. Minegishi, Y., E. Coustan-Smith, L. Rapalus, F. Ersoy, D. Campana, and M.E. Conley. 1999. Mutations in Iga (CD79a) result in a complete block in B-cell development. J. Clin. Invest. 104:1115–1121.[Medline]
20. Minegishi, Y., J. Rohrer, E. Coustan-Smith, H.M. Lederman, R. Pappu, D. Campana, A.C. Chan, and M.E. Conley. 1999. An essential role for BLNK in human B cell development. Science. 286:1954–1957.[Abstract/Free Full Text]
21. Ochs, H.D., and C.I. Smith. 1996. X-linked agammaglobulinemia. A clinical and molecular analysis. Medicine (Baltimore). 75:287–299.[CrossRef][Medline]
22. Gong, S., and M.C. Nussenzweig. 1996. Regulation of an early developmental checkpoint in the B cell pathway by Ig beta. Science. 272:411–414.[Abstract]
23. Ferrari, S., R. Zuntini, V. Lougaris, A. Soresina, V. Sourkovà, M. Fiorini, S. Martino, P. Rossi, M.C. Pietrogrande, B. Martire, et al. 2007. Molecular analysis of the pre-BCR in a large cohort of Italian patients affected by autosomal recessive agammaglobulinemia. Genes Immun. 8:325–333.[CrossRef][Medline]
24. Costa, T.E., R.R. Franke, M. Sanchez, Z. Misulovin, and M.C. Nussenzweig. 1992. Functional reconstitution of an immunoglobulin antigen receptor in T cells. J. Exp. Med. 175:1669–1676.[Abstract/Free Full Text]
25. Han, K., Y. Kim, J. Lee, J. Lim, K.Y. Lee, C.S. Kang, W.I. Kim, B.K. Kim, S.I. Shim, and S.M. Kim. 1999. Human basophils express CD22 without expression of CD19. Cytometry. 37:178–183.[CrossRef][Medline]
26. Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, and K.A. Rajewsky. 1992. A critical role of lambda 5 protein in B cell development. Cell. 69:823–831.[CrossRef][Medline]
27. Wossning, T., and M. Reth. 2004. B cell antigen receptor assembly and Syk activation in the S2 cell reconstitution system. Immunol. Lett. 92:67–73.[CrossRef][Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This Article Abstract Full Text (PDF) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JEM Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ferrari, S. Articles by Plebani, A. Search for Related Content PubMed PubMed Citation Articles by Ferrari, S. Articles by Plebani, A. Social Bookmarking                      What's this?