B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection

Abstract

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS. Cryosections from an AS-derived SM were stained for B and T lymphocytes. B cells were isolated from different areas of a focus. The rearranged VH genes were amplified by semi-nested polymerase chain reaction (PCR) using oligonucleotides specific for the six different VH families and heavy chain joining segments (JHs). PCR products were cloned and sequenced. Fifty-nine of 70 different heavy chain gene rearrangements were potentially functional. Most of the rearranged genes were mutated (range, 1–15%). Thirty of 70 products had a mutational pattern typical for antigen selection. Most of the rearranged VH genes belonged to the VH3 family (54%), consistent with data from healthy donors and patients with RA, while VH4 genes, in contrast to RA, were identified less frequently (10%) and VH5 genes were over-represented (11%). In contrast to RA, neither VH6 genes nor the autoimmunity-prone VH4-34 were seen, whereas another autoimmunity-prone gene, V3-23, was predominantly used (11%). One VH1-derived and one VH3-derived B cell clone were expanded. CDR3 were shorter and more variable in length than in RA. Comparable with RA and reactive arthritis, there is a biased repertoire of selected VH genes, whereas the panel of represented genes is different and less clonal expansion was observed.

Introduction

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]. The B cell subset in AS joints has not yet been characterized. We analyzed the repertoire and distribution of rearranged VH genes to elucidate B lymphocyte involvement in AS, and to investigate similarities to and differences from RA.

Tissue preparation and staining

]. The stained B lymphocytes from serial sections taken at 50μm intervals were isolated by microdissection, and DNA preparation followed (see supplementary material).

VH PCR

). A similar PCR without DNA was run as a negative control.

Template generation and sequence analysis

PCR products of the expected 350 base pair (bp) length were detected by standard agarose gel electrophoresis and purified. The purified DNA template was bacterially subcloned followed by plasmid isolation and sequencing. Sequences were analyzed by homology comparison with the EMBL and GenBank gene databases (see supplementary material).

The VH gene usage

Except for the negative control, all VH PCRs led to a product of the expected length (350 bp). All sequences reported in the present paper are accessible on the EMBL database.

).

Twelve genes in all were nonfunctionally rearranged, with the highest proportion in VH3 (18%), about 14% in VH1, VH4 and VH5, and none in VH2.

VH1 genes

Fourteen individual rearrangements represented VH1 genes. The most often represented VH1 gene was DP75 (four products) followed by DP10, DP14, DP21 and DP25 (two products each). Out of 14 distinct VH1–DH–JH sequences, 2 were nonfunctional (14%). CDR3 lengths varied between 18 and 39 bp (mean, 28.5 bp).

). With 99% homology to DP21, they shared three mutations within the framework region (FR).

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VH2 genes

Three different VH2 rearrangements were determined. All were potentially functional and > 95% homologous to the respective gl gene. The number of mutations ranged from 2 (5c-VH22s) to 10 (5c-VH26s). R:S ratios were quite low, with a maximum of 2.0 in the CDR (5c-VH26s). CDR3 lengths varied between 15 and 45 bp (mean, 32 bp).

VH3 genes

Thirty-eight different VH3 rearrangements were characterized: eight were most homologous to DP47/V3-23 (21%), five to DP50 (13%), four each to DP54 and DP31 (11%), respectively, and three each to DP49, DP35, DP51 and DP77 (8%), respectively. Seven (18.4%) VH3 genes were nonfunctionally rearranged. CDR3 lengths varied from 6 to 35 bp (mean, 27.2 bp).

). These genes, with 95.5% homology to DP54, shared four replacement mutations, and six mutations each were localized at different positions.

Twenty-three of the VH3 sequences were highly mutated (10–27 mutations). Except for one case, most of the mutations were localized in the CDR with R:S ratio >3. Three of seven nonfunctionally rearranged genes also revealed a pattern of antigen-induced mutation.

VH4 genes

Seven different VH4 rearrangements could be characterized; six were potentially functional. CDR3 lengths ranged from 12 to 45 bp (mean, 28 bp).

The gl genes HUMIGHCAK, HUMIGHCAG and DP63 were represented twice each, and DP71 once. No homology to DP64/VH4-34/VH4.21 was seen. Five of the sequences had few mutations (one to eight) with a R:S ratio higher in the FR than in the CDR. Two VH4 rearrangements had a R:S ratio >3 in the CDR (5c-VH44a and 3b-VH45a).

VH5 genes

Eight distinct VH5 rearrangements could be determined; seven were potentially functional. CDR3 lengths ranged from 12 to 66 bp (mean, 39.8 bp).

The 3a-VH54s gene was unmutated. There were 7–35 mutations in all other sequences. For five sequences (four were potentially functional), the mutational pattern revealed antigen induction.

VH PCR

], a comparison is possible.

The VH gene usage

], was not detected within the VH4 genes from AS SM.

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Clonal expansion

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The CDR3

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Mutational pattern

Only nine rearrangements were not mutated. R:S ratios >3 within the CDR were observed in 30 genes (two were nonfunctional). Members of the VH1, VH3, VH4 and VH5 families carried more somatic mutations than the VH2 genes.

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Nonfunctional rearrangements

]. In contrast, no bias to elevated CDR3 lengths was seen.

Conclusion

An active immune reaction with GC formation occurs in the inflamed SM in AS. B cell affinity maturation with generation of somatically mutated antibody-coding genes characteristic for memory cells occurs in these GC formations, indicating an antigen-driven response. Comparable with RA and ReA, there is a biased repertoire of selected VH genes, whereas the panel of represented genes is different and clonal expansion was observed less frequently.

Introduction

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DNA preparation

O. The sample was incubated over 45 min at 55°C. After inactivation of proteinase (95°C), 25 μl of the solution was subjected to the first PCR.

Seminested VH PCR

First PCR. An oligonucleotide mix of VH1, VH2, VH3, VH4, VH5, VH6 and an oligonucleotide mix of JH-Intron 1,2-4,5 and JH-Intron 3,6 as primers (final concentration, 0.125 μM each), 200 μM dNTP, 2 mM MgCl2, 1 U Goldstar Taq-polymerase (Eurogentec, Seraing, Belgium), and the manufacturer's reaction buffer. First cycle: 5 min denaturation at 95°C, 3 min annealing at 58°C and 90 second extension at 72°C; cycles 2–35, 80 second denaturation, 30 second annealing, and 90 second extension, with a final extension of 5 min.

Second PCR. One microliter of the product of the first PCR with individual oligonucleotide primers VH1–VH6 and JH1–JH6, under the same conditions as the first PCR except for annealing for VH1, VH2, VH5, VH6 at 58°C, and for VH3 and VH4 at 63°C.

DNA purification and plasmid ligation were performed with commercial kits following the manufacturer's instructions: DNA purification from the agarose-gel with the QUIAquick kit (Quiagen, Hilden, Germany), and bacterial cloning with the TA-cloning kit (Invitrogen, Leek, The Netherlands).

For DNA sequence homology search and sequence comparison, DNASIS software (Hitachi Europe, Olivet, France), and EMBL Nucleotide Sequence Submissions (European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK) were used. The GenBank (National Institutes of Health, Bethesda, MD, USA) was also used.

Abbreviations

AS = ankylosing spondylitis; CDR = complementarity determining region; DH = heavy chain diversity segment; FR = framework region; GC = germinal centers; gl = germline; JH = heavy chain joining segment; PCR = polymerase chain reaction; RA = rheumatoid arthritis; ReA = reactive arthritis; SM = synovial membrane; VH = heavy chain variable segment.

Acknowledgement

This work was supported by Deutsche Forschungsgemeinschaft Ga320, 3-1.

Figures and Tables

The synovial focus. Double-immunohistological staining (anti-CD20 alkaline phosphatase-anti-alkaline phosphatase and anti-CD3 streptavidin biotin horseradish peroxidase reaction) of 8 μm sections from frozen SM obtained by total joint replacement of an AS patient's right hip (B cells, red staining; T cells, brown staining).

Distribution of the VH families among the PCR products obtained from different slides (2c, 3a, 3b and 5c). Bars for each section represent the respective number of products for each indicated VH family.

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VH3 in comparison with their respective gl gene. The amino acid position and the CDR positions are marked. Dashes indicate identity to the corresponding character on top. Amino acid replacements are indicated in the last line.

The rearranged heavy chain variable segment genes, their functionality, respective germline gene (homology), ratio of mutations leading to amino acid replacement to silent mutations with the number of mutations for the complementarity determining region and the framework region, heavy chain joining segments and CDR3 lengths

Identical to F5-3a4, F5-3b4, 5c-4m6.

Keywords

• ankylosing spondylitis
• B lymphocyte immunology
• heavy chain genes
• immunoglobulins
• somatic mutations