Statistical significance was determined using one-way ANOVA with Tukeys post test. plasma cellspecific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation. == INTRODUCTION == Memory B cells (Bmem) and plasma cells (PCs) serve a key function in providing long-lasting humoral protection to pathogenic challenge, both in the context of natural infections and following vaccinations (1,2). Despite these central roles, many questions remain unanswered about these cells, including the functions of subpopulations of human Bmem and PC, regulatory mechanisms governing their activation, and the basis of their longevity. One impediment to answering these questions is the paucity of suitable markers for specific Bmem identification. Blood Bmem in humans are identified by the expression of CD27 (3,4), whereas tissue-based Bmem are recognized as CD19+/IgD/CD38(5,6). Although widely used as a marker for Bmem, CD27 is also expressed on germinal center (GC) B cells, PC, and many nonB lineage cells (7,8), and several studies identified Bmem populations lacking CD27 expression (911). Consequently, Bmem are currently best defined as antigen-experienced B cells, which are not engaged in an ongoing immune reaction, rather than described by the expression of well-defined cell surface markers. To address this issue, we developed a structure-based approach for the identification of cell surface antigens on Bmem using the variable lymphocyte receptor (VLR) antibodies of the jawless vertebrate sea lamprey (Petromyzon marinus). Despite observations of adaptive immune responses by jawless vertebratesthat is, lampreys and hagfishover 50 years ago (12,13), no homologs of conserved prototypical adaptive immune genes, such as human leukocyte antigen (HLA) or recombining B cell and T cell antigen receptors, could be identified in evolutionarily distant jawless vertebrates. Only recently, the molecular basis of adaptive immune responses in jawless vertebrates was defined with the discovery of clonally diverse VLR anticipatory receptors (14). This receptor system provides a potential repertoire exceeding 1014clonotypes (1416). Unlike antibodies of jawed vertebrates, which use the immunoglobulin (Ig) fold as the basic structural unit, the VLRB antibodies consist of sheetforming leucine-rich repeat (LRR) units. Structural analyses of monoclonal Lazabemide VLRB antibodies in complex with protein and carbohydrate antigens indicate that residues of VLRB antibodies located on the inner concave surface and a variable loop structure emanating Lazabemide from the C-terminal capping LRR unit interact with the antigen (1719). We reasoned that Lazabemide the radically different protein architecture of VLR antibodies and the large evolutionary distance of jawless vertebrates from jawed vertebrates would allow the generation of monoclonal VLR antibodies to antigens, which conventional mammalian antibodies do not readily recognize because of structural or tolerogenic constraints. In previous studies, we established monoclonal VLR antibodies as a new class of research reagents for discovery of biomarkers on human lymphocytes and PCs (20,21). Here, we report the isolation of the monoclonal antibody VLRB N8, reactive with an epitope that is expressed specifically on HLA-I on Bmem and PC, is up-regulated on Bmem in autoimmune disorders and is dependent on tyrosine sulfation of HLA-I, indicating that HLA-Idependent immune responses are subject to an as yet unexplored level of immune regulation. == RESULTS == == VLRB N8 specifically recognizes human Bmem and PCs == In an effort to generate novel CLEC10A reagents targeting late stages in B lineage differentiation, we screened 628 monoclonal VLRB antibodies from a library generated from lymphocytes of sea lamprey larvae immunized with a cocktail of the plasmacytoma cell lines NCI-H929, U266, and RPMI 8226. Monoclonal VLRB antibodies that displayed reactivity to these cell lines were further evaluated for recognition of primary lymphocytes. One of these VLRB antibodies, VLRB N8, recognized human CD27+/IgDBmem, and CD27+/IgD+marginal zone equivalent (MZe) cells.