application of scfv-phage display to analysis of b-cell...

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Original article

Application of scFv-phage display to analysisof B-cell clones proliferating in the salivaryglands of a patient with Sjogren’s Syndrome

D.I. Stott and G.P. SimsDept. of Immunology, University of Glasgow, WesternInfirmary, Glasgow G11 6NT, Scotland, UK

1. Introduction

Sjogren’s Syndrome (SS) is an autoimmune diseasecharacterized by chronic B- and T-cell infiltration of thesalivary and lachrymal glands. Clusters of B- and T-cells which resemble germinal centres are seen withinthe glandular tissue. We have shown that clonal B-cell proliferation and affinity maturation are occurringwithin these clusters [1]. ScFv antibodies have beenproduced from one of these B-cell clusters to identifythe autoantigen that induces a germinal centre reactionand study the effects of somatic hypermutation on theautoimmune response.

2. Methods

Frozen sections of a lip biopsy from a patient withSS were stained with monoclonal antibodies to detectB-cells, T-cells, follicular dendritic cells and plasmacells using the APAAP technique (Fig. 1(a)–(d)). B-cell clusters were excised from the tissue by microdis-section and the rearranged immunoglobulin V-genesamplified by PCR, cloned and sequenced [1]. Thebest matching germline V-gene was identified for eachheavy and light chain V-gene sequence. The num-ber of somatic mutations and ratio of replacement tosilent mutations (R/S ratio) was determined. V-genesderived from members of the same B-cell clone wereidentified by their use of the same germline V, D, J

and CDR3 sequence. Clonally related VH and VL

genes were used to construct scFvs using a (Gly4Ser)3linker. The scFv construct was cloned using phagemidpCANTAB6 (Cambridge Antibody Technology) andexpressed as phage-displayed scFv-gp3 fusion proteinor soluble scFv [2].

3. Results

Clusters of lymphocytes in the salivary gland biop-sies were shown by immunohistology to contain B-cells (CD20+), T-cells (CD3+) and follicular dendriticcells (Fig. 1(a)–(c)). The clusters were surrounded byplasma cells (Fig. 1(d)). This resembles the cellularcomposition of a germinal centre, so we excised clus-ters from 8 micron sections and cloned the rearrangedIg V-genes. A single, clonally related set of VH-geneshomologous with the germline gene DP-10, and a clon-ally related set of Vλ-genes, homologous with DPL16,were isolated from one cluster (Table 1). The onlyother clonally related set was homologous with VH -DP65 and all members of this set were non-functional.We therefore concluded that DP-10 and DPL16 wereexpressed by the same dominant B-cell clone present inthis cluster. The ratio of replacement/silent mutationsdeviated significantly from random (2.9), indicatingthat the clone had been subjected to antigen selection.

Genealogical analysis of the relationships betweenthe sequences allowed us to construct family trees onthe basis of somatic mutations in the V-genes, showingthat the B-cell clone had proliferated in the salivarygland (Fig. 2). A mini-library of phage-displayed scFvswas constructed from these VH and VL-genes. Noneof the scFvs bound specifically to Ro, La or IgG byELISA or immunoblotting. We are using this library tosearch for other salivary gland antigens that might bedriving the B-cell response.

Disease Markers 16 (2000) 21–23ISSN 0278-0240 / $8.00 2000, IOS Press. All rights reserved

22 D.I. Stott and G.P. Sims / Application of scFv-phage display to analysis of B-cell clones

Fig. 1. Phenotypes of a cell cluster in a labial gland biopsy from a patient with Sjogren’s syndrome. Sequential sections stained with monoclonalantibodies to (a) CD20 (B-cells), (b) CD3 (T-cells), (c) Follicular dendritic cells, (d) Plasma cells.

Table 1Rearranged Ig V-genes isolated from a cell cluster in a labial salivary glandbiopsy of a Sjogren’s syndrome patient were identified with the best matchinggermline V-gene in the human Vbase Sequence Directory (Tomlinson et al.,Cambridge, UK). The cluster contains a dominant proliferating B-cell cloneexpressing VH -gene DP10 and VL-gene DPL16 which have undergone hyper-mutation

V-gene Germline No. of Av. No. of Mean R/S± S.D.family V-gene sequences mutations/ Framework CDRs

isolated V-region region

VH1 DP-10 21 14.7± 1.2 1.2± 0.22 0.9± 0.19VH4 DP-65a 8Vκ1 L5 2 9 3.0 4.0Vκ1 08/018a 1Vκ4 B3 2 1.5± 0.5 > 1 > 0.5

B3a 1Vκ5 B2a 1Vκ3 DPL16 10 8.6± 1.1 1.2± 0.24 > 4 ± 0.6

DPL16a 1 10 1.0 > 4aNon-functional.

4. Conclusions

Cell clusters present in labial biopsies from SS pa-tients contain B-cells, T-cells and follicular dendriticcells, and the surrounding glandular tissue is packedwith plasma cells. This is characteristic of germinalcentres found in secondary lymphoid organs undergo-ing an immune response. The rearranged Ig V-genesequences expressed by cells in the cluster describedhere indicate that the majority of B-cells are derived

from a single dominant clone that has proliferated inthe target tissue. Analysis of the sequences shows thatthis process is driven by somatic hypermutation of theIg V-genes and antigen selection. ScFvs were con-structed from a mini-library of VH and VL genes fromthis B-cell clone and used to identify which autoanti-gen is driving the immune response. Phage-displayedscFvs and soluble scFv are being tested against a panelof potential antigens using ELISA and western blot-ting. This technique can be used to identify novel anti-

D.I. Stott and G.P. Sims / Application of scFv-phage display to analysis of B-cell clones 23

Fig. 2. Genealogical trees of the dominant clonally related VH and VL-genes. (a) VH DP-10 clone, (b) VL DPL16 clone. Numbers adjacent tothe arrows show the number of point mutations.

gens driving the immune response target tissues and tostudy the relationship between somatic hypermutationand affinity maturation. So far as we are aware, this isthe first time Ig V-genes, isolated from a B-cell clonewithin the target tissue during an immune response,have been used to reconstruct the original antibodiesand determine the nature of the antigen driving the re-sponse.

Acknowledgements

The pCANTAB6 vector was kindly provided by

Cambridge Antibody Technology. This work wasfunded by an EC Marie Curie Research Fellowship andby the MacFeat Bequest, University of Glasgow, Scot-land, UK.

References

[1] D.I. Stott, F. Hiepe, M. Hummel, G. Steinhauser and C.Berek,J. Clin. Invest. 102 (1998), 938–946.

[2] A.R. Pope, M.J. Embelton and R. Mernaugh, in:Antibody En-gineering, J. McCafferty, H.R. Hoogenboom and D.J. Chiswell,Oxford University Press, UK, 1996, pp. 1–40.

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