When this method was applied in patients with known immunizing histories (i.e; kidney allograft recipients with circulating antibodies against HLA antigens), a significant increase in the frequency of B cells specific to HLA class I molecules was observed. the characterization of antigen-specific blood B cells. Introduction The crucial role of B cells in a number of autoimmune diseases, such as multiple sclerosis [1] and rheumatoid arthritis [2], has been recently highlighted through the study of anti-CD20 in clinic. Having access to specific antigen committed blood B cells in humans would be an important step towards better understanding B cells potential role in autoimmunity and responses against infectious agents and allotransplants. B cells are not only plasmocyte progenitors, but CDK2-IN-4 also display regulatory functions [3], [4], are good presenting cells [5] and can have direct cytotoxic effects[6]C[8]. Mechanisms shaping the early B cell repertoire rely predominantly on receptor editing and anergy, and not on deletion [9], [10]. However, in humans a substantial frequency of mature circulating B cells still show some degree of autoreactivity and or polyreactivity, which survives the first checkpoint of B cell repertoire maturation [11], and persisting autoreactive B cells in the mature repertoire [12]. There is thus a continuous need for effective regulation C mostly from TREGC to avoid any deleterious reaction. In human, the analysis of autoreactive B cell frequency has been most often indirectly approached using the reactivity of antibodies produced in B cell culture supernatants in limiting dilution conditions [13], where it seems that tools identifying committed B cells by direct interaction would be more effective. A number of such direct interaction approaches have been developed such as the use of modified tetramers that consist of a R-PE-labeled streptavidin core and four biotinylated proteins [14]. The main limitation of such an approach is the heterogeneous binding of B cells. B cells will not only bind to the target protein but also to the fluorescent molecule (i-e PE) and biotin epitopes within the tetramer. To circumvent this problem, a concomitant use of another tetramer (conjugated to a different fluorochrome) is needed to exclude unspecific binding. In addition, such a method may face technical difficulties in achieving a stereotyped labeling of the reagents, which may vary from batch to batch. In this report, we used fluorescent Bio-plex COOH beads that contain a fluorescent internal core and can be covalently linked to any protein. A broad variety of antigens can be analyzed simultaneously through varying the ratio of two fluorescent molecules within the bead internal core. The strategy was first assessed using B cells purified from 8.18-C5 transgenic mice expressing human anti-MOG BCR [15]. B cells purified from healthy human blood and immunized individuals were then tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, all of which may have some usefulness to the study of various pathological processes. For instance, we show increased frequencies of anti HLA committed B cells in patients with CDK2-IN-4 circulating anti HLA CDK2-IN-4 antibodies compared to unsensitized patients or normal individuals. We also show that, similarly to T cells [16], [17], a substantial amount of B Rabbit Polyclonal to PTGDR cell binding self-antigen MOG coated beads can be detected in normal individual blood, confirming the permissivity of the first B cell tolerogenic checkpoint in humans. Furthermore we.