Scholarlink

Objective <p>Several autoimmune diseases are characterized by the presence of autoantibodies to nuclear antigens. They are present in essentially all patients with systemic lupus erythematosus (SLE). The study of antinuclear antibodies (ANA) produced by individual B cells can offer critical insights into the structural and functional characteristics of autoantibodies. Conventional techniques of single-cell cloning and sequencing or single-cell (Nojima) cultures have significant limitations. To overcome these limitations, we developed an integrated platform that combines flow cytometric detection of ANA-reactive B cells with an optimized single-B cell culture system.</p> Methods <p>We employed a flow cytometry assay using biotinylated nuclear extracts and fluorochrome-tagged streptavidin to detect ANA-reactive B cells, followed by single cell sorting and culture in the presence of feeder cells expressing CD40L and BAFF, IL-2 and IL-21. This integrated approach was designed to enhance clonal resolution while increasing the efficiency of ANA-specific B cell recovery compared to conventional single-cell cloning methods.</p> Results <p>We isolated and expanded ANA + and ANA- B cells of various B cell subsets (naive, CD27 + IgM + , and IgG memory) that were isolated from healthy donors and patients with SLE. The cultures were maintained in standard conditions for 26&#xa0;days at which time 82% of naïve, 53% of CD27 + IgM + and 64% of IgG⁺ memory B cells produced &gt; 1.0 ug/mL of immunoglobulin. We further demonstrated that 88—95% of ANA + B cells Ig produced ANA-reactivity by HEp-2 staining.</p> Conclusions <p>This method establishes a robust platform for isolating and expanding ANA B cells, facilitating the production of monoclonal autoantibodies and analysis of B cell receptor (BCR) gene sequences. This expansion is achieved by culturing single ANA + B cells with CD40L-expressing feeder cells and key cytokines (BAFF, IL-2, and IL-21) which together promote their survival, proliferation, and differentiation into antibody-secreting cells. The approach offers important new potential in understanding the etiology of ANA- reactive IgG in autoimmune disease by enabling the identification of specific autoantigen targets at distinct stages of B cell differentiation.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

An efficient approach to study ANA⁺ B cells in autoimmune diseases integrating flow cytometry with single-cell analysis

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年5月18日

查看全文
  • Muriel Velez Arteaga,
  • Shabirul Haque,
  • Manami Watanabe,
  • Yemil Atisha-Fregoso,
  • Sophia Garcia,
  • Cynthia Aranow,
  • Meggan Mackay,
  • Bruce T. Volpe,
  • Betty Diamond

摘要

Objective

Several autoimmune diseases are characterized by the presence of autoantibodies to nuclear antigens. They are present in essentially all patients with systemic lupus erythematosus (SLE). The study of antinuclear antibodies (ANA) produced by individual B cells can offer critical insights into the structural and functional characteristics of autoantibodies. Conventional techniques of single-cell cloning and sequencing or single-cell (Nojima) cultures have significant limitations. To overcome these limitations, we developed an integrated platform that combines flow cytometric detection of ANA-reactive B cells with an optimized single-B cell culture system.

Methods

We employed a flow cytometry assay using biotinylated nuclear extracts and fluorochrome-tagged streptavidin to detect ANA-reactive B cells, followed by single cell sorting and culture in the presence of feeder cells expressing CD40L and BAFF, IL-2 and IL-21. This integrated approach was designed to enhance clonal resolution while increasing the efficiency of ANA-specific B cell recovery compared to conventional single-cell cloning methods.

Results

We isolated and expanded ANA + and ANA- B cells of various B cell subsets (naive, CD27 + IgM + , and IgG memory) that were isolated from healthy donors and patients with SLE. The cultures were maintained in standard conditions for 26 days at which time 82% of naïve, 53% of CD27 + IgM + and 64% of IgG⁺ memory B cells produced > 1.0 ug/mL of immunoglobulin. We further demonstrated that 88—95% of ANA + B cells Ig produced ANA-reactivity by HEp-2 staining.

Conclusions

This method establishes a robust platform for isolating and expanding ANA B cells, facilitating the production of monoclonal autoantibodies and analysis of B cell receptor (BCR) gene sequences. This expansion is achieved by culturing single ANA + B cells with CD40L-expressing feeder cells and key cytokines (BAFF, IL-2, and IL-21) which together promote their survival, proliferation, and differentiation into antibody-secreting cells. The approach offers important new potential in understanding the etiology of ANA- reactive IgG in autoimmune disease by enabling the identification of specific autoantigen targets at distinct stages of B cell differentiation.