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SAT0183 Clonal B cell populations in renal tissue of patients with autoimmune nephropathy: Detection by spectratyping analysis
  1. P.P. Sfikakis1,
  2. S. Kolovou2,
  3. S. Marinaki2,
  4. M. Roumelioti1,
  5. D. Paleologou1,
  6. J. Boletis2,
  7. P. Panayiotidis1
  1. 1First Department of Propaedeutic and Internal Medicine, Athens University Medical School
  2. 2Nephrology Department, Laikon Hospital, Athens, Greece

Abstract

Background We have previously shown that clusters of clonaly-related immunoglobulin heavy-chain (Ig-VH/DH/JH) sequences are commonly found in the peripheral blood of patients with active lupus nephritis using PCR, cloning, sequencing and analysis of >1500 rearranged Ig-VH/DH/JH genes. In addition to the original somatic mutation(s) shared by all clonally-related sequences, the presence of additional ongoing mutations indicated expansion of single B-cell precursors and clonal evolution, implying a preferential survival/growth of potentially autoreactive B-cells [1]. Whether clonal B cell populations are present in renal tissue, induced as a result of specific autoantigen(s) that locally exist, is not known.

Objectives To search for clonal B cell populations in the renal tissue of patients with autoimmune nephropathies using VH multiplex PCR and spectratyping analysis.

Methods Genomic DNA extracted from renal biopsy samples was subjected to multiplex PCR for the detection of Ig-VH clonal rearrangement using the IGH Gene Clonality Assay (InVivoScribe Technologies, Netherlands). Each DNA sample (200ng μg) was subjected to PCR three times using FR1, FR2 and FR3 multiplex PCR primer sets according to the BIOMED 2 Concerted Action protocol (BMH4-CT98-3936). PCR products were analyzed by capillary electrophoresis (Beckman CEQ 6000) and PCR fragments were analysed according to the Biomed-2 guidelines for the definition of the presence of clonal B cell populations. Results are expressed as presence of either a) polyclonal, b) oligoclonal, or c) monoclonal B cell populations in the analyzed DNA from the renal tissue examined.

Results Electrophoregrams of PCR products derived from renal tissue of 7 patients with biopsy proven active autoimmune nephropathies (lupus nephritis, n=5; membranous nephropathy, n=2) revealed the presence of a monoclonal B cell population among polyclonal B cells in 2 (lupus nephritis class III and membranous nephropathy), B cell oligoclonality in 3, whereas polyclonal B cells were found in the remaining 2 patients. In contrast, clonal B cell populations were not found in any of 8 control renal samples obtained either at surgery from patients with renal cancer (n=6) or derived from biopsies of patients with non-autoimmune nephropathies (diabetic nephropathy and thin membrane disease).

Conclusions These preliminary results indicate that clonaly expanded B cells in the renal tissue detected by spectratyping analysis may serve as a specific biomarker of autoimmune nephropathy and may identify individual patients who could benefit from therapeutic B cell depletion. Further studies to identify the VH sequence(s) of clonal B cells that could bind specific (auto)antigens expressed in renal tissue of these patients are underway.

  1. Sfikakis P.P. et al. Clin Immunol. 2009;1:19-31.

Disclosure of Interest None Declared

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