Elucidation of the mechanism behind this observation could be of relevance to the identification of those citrullinated antigens that drive ACPA responses and could provide clues to how the continuous recruitment of new B cells can be halted. Abbreviations ACPA: anticitrullinated protein antibody; Arg: arginine; AU: arbitrary unit; BL: baseline; Cit: citrulline; CCP2: cyclic citrullinated peptide 2; EAC: Early Arthritis Medical center; ELISA: enzyme-linked immunosorbent assay; Fib: fibrinogen; FU: follow-up; Ig: immunoglobulin; IgM: immunoglobin M LUMC: Leiden University or college Medical Center; MBP: myelin basic protein; RA: rheumatoid arthritis; RF: rheumatoid factor. Competing interests The authors declare that they have no competing interests. Authors’ contributions PS, TWJH, REMT and LAT conceived the study and participated in its design and edited the manuscript. controls. IFI35 Results Acknowledgement of defined citrullinated antigens by IgM Trimetrexate ACPA was confined to samples that also displayed acknowledgement by IgG ACPA. However, the IgM ACPA response displayed a more restricted antigen acknowledgement profile than IgG ACPA (OR = 0.26, P < 0.0001). Conclusion Our data show that several defined citrullinated antigens are acknowledged only by IgG ACPA, whereas others are also recognized by IgM ACPA. These observations suggest that not all citrullinated antigens are able to activate new B cells despite concurrent acknowledgement by Trimetrexate IgG ACPA. Introduction Anti-citrullinated protein antibodies (ACPA) may be involved in the disease pathogenesis of rheumatoid arthritis (RA). ACPA can be found early in the disease course [1], even before disease onset [2], and the presence of ACPA at the time of diagnosis can predict disease course [3]. Moreover, ACPA can contribute to disease pathogenesis by activating immune cells [4,5] and the match system [6]. The ACPA response likely represents a T-cell-dependent B-cell response, given the protein nature of the antigen acknowledged and the strong association with the human leukocyte antigen shared epitope alleles. The development of such a response is typically characterized by a first wave of IgM antibodies after the first antigen contact, quickly followed by the Trimetrexate presence of IgG. After repeated antigen exposure, the IgG responses are further boosted while the IgM peak declines. The latter observation is explained by the presence of Ig-switched, affinity matured, memory B cell that are created in the presence of CD4+ T cells. These T cells provide the helper activity required for affinity maturation, isotype switching and memory cell formation. When such T-cell help cannot be provided, as in the case of hyper-IgM syndrome, IgG, IgA and IgE antibody levels are absent or severely reduced [7]. The presence of IgG, IgA and IgE ACPAs [5,8], therefore, provides another line of evidence for the T-cell-dependent nature of ACPA responses. To the best of our knowledge, IgM-producing memory B cells against T-cell-dependent antigens have not been described, in contrast to T-cell-independent B-cell responses against, for example, repetitive sugar residues on bacteria [9,10]. For these reasons, it is most conceivable that the presence of IgM ACPA suggests that activation of recently recruited na?ve B cells recognize citrullinated antigens because the half-life of circulating IgM is short. In this study, we hypothesized that there might be certain antigens which drive the ACPA IgM response in RA. We therefore sought to determine whether there is a difference in the fine specificity of IgG and IgM ACPA. Materials and methods Fine specificity of anticitrullinated protein antibody immunoglobulin M We determined the fine specificity of ACPA IgM and IgG in 113 serum samples of anti-cyclic citrullinated peptide (CCP2) IgG and IgM double-positive RA patients collected from the Leiden Early Arthritis Clinic (EAC), an inception cohort of recent-onset arthritis that was initiated at the Department of Rheumatology at Leiden University Medical Center in 1993 [11]. We selected those patients who had a relatively high titer of IgM CCP2 (levels 100 AU/ml) to ensure that differences between IgG and IgM reactivity could not be explained by differences in the sensitivity associated with the detection of IgG or IgM antibodies by ELISA. For the determination of ACPA status and ACPA levels of IgM anti-CCP2, we used a commercial immunoassay kit (Euro-Diagnostica, Malm?, Sweden) with minor modifications for the detection of IgM. The collection and use of patient samples was approved by the local medical ethics committee and in compliance with the Declaration of Helsinki. All patients provided their written informed consent. Demographic data (age, gender, disease duration and radiographic damage) of ACPA IgG-positive RA patients who.