Isolation and Characterization of Anti-Citrullinated Protein Antibody-Secreting Cells in Rheumatoid Arthritis

Plasma cells were obtained from the synovial fluid of a patient with anti‐CCP–positive RA, and antibody‐secreting cells were isolated from synovial fluid mononuclear cells using the fluorescent foci method. In addition, single citrulline‐specific B cells were sorted by flow cytometry from the peripheral blood of other RA patients, using an antigen‐tetramer system. Further details on the synovial fluid and serum samples obtained from RA patients and the methods used for plasma cell isolation, blood‐derived memory B cell tetramer isolation, Ig gene sequence analysis, cloning of Ig genes, generation of germline‐reverted antibodies, expression and purification of monoclonal antibodies (mAb), surface plasmon resonance (SPR) assay, ACPA peptide array, PTM peptide enzyme‐linked immunosorbent assay (ELISA), in solution citrullination ELISA, immunoprecipitation and osteoclast cultures, and in vitro bone erosion assay are provided in Supplementary Materials and Methods (available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40699/abstract).

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Steen J., Forsström B., Sahlström P., Odowd V., Israelsson L., Krishnamurthy A., Badreh S., Mathsson Alm L., Compson J., Ramsköld D., Ndlovu W., Rapecki S., Hansson M., Titcombe P.J., Bang H., Mueller D.L., Catrina A.I., Grönwall C., Skriner K., Nilsson P., Lightwood D., Klareskog L, & Malmström V. (2019). Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell–Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis. Arthritis & Rheumatology (Hoboken, N.j.), 71(2), 196-209.

Publication 2019

Anti ccp Antibodies Antibody secreting cells Antigen Arthritis Assay B cells Blood Bone Cell isolation Citrullination Citrulline Enzyme linked immunosorbent assay Flow cytometry Germline Ig genes Immunoprecipitation Isolation Memory b cell Monoclonal antibodies Osteoclast Patients Peptide Plasma Plasma cells Sequence analysis Serum Surface plasmon resonance Synovial cells Synovial fluid Tetramer

Corresponding Organization : Karolinska Institutet

Other organizations : KTH Royal Institute of Technology, Charité - Universitätsmedizin Berlin, UCB Pharma (United Kingdom), Thermo Fisher Scientific (Sweden), Uppsala University, University of Minnesota, University of Minnesota Medical Center

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Variable analysis

independent variables

Isolation of antibody-secreting cells from synovial fluid mononuclear cells using the fluorescent foci method
Sorting of single citrulline-specific B cells from the peripheral blood of other RA patients using an antigen-tetramer system

dependent variables

Antibody secretion by plasma cells
Binding of antibodies to citrullinated antigens (ACPA peptide array, PTM peptide ELISA, in solution citrullination ELISA)
Ability of antibodies to induce osteoclast differentiation and bone erosion (immunoprecipitation, osteoclast cultures, in vitro bone erosion assay)

control variables

Synovial fluid and serum samples obtained from RA patients

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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