Profiling of B cell subsets over the vaccination program revealed that KLH-specific B cells matured from nave to class-switched memory space B cells, confirming the prototypic B cell response to a neo-antigen. cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is definitely specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of fresh flow-cytometric protocols. Keyhole limpet hemocyanin (KLH) is a high-molecular-weight glycoprotein of marine source that induces both cell-mediated and humoral reactions in animals and humans. Because of its potent immunogenicity, its low-grade toxicity and its availability like a medical grade product, KLH is used extensively as a natural immunostimulant for basic research and medical Methoctramine hydrate applications1,2,3. Like a neo-antigen, KLH is definitely ideally suited to study T cell-dependent main and secondary immune responses and a recent study shows its ability to activate the innate immune system. KLH was first introduced into the medical center in 1967 to assess immunocompetence of individuals4. KLH is currently mainly used as standard carrier protein for the production of monoclonal antibodies to haptens such as peptides and oligosaccharides1. Besides this, KLH has been studied Methoctramine hydrate as a local treatment for individuals with bladder malignancy, but proved to be inferior to mitomycin Methoctramine hydrate treatment5,6. Finally, KLH offers progressed into medical trials as either a carrier protein, an adjuvant- or immunomonitoring tool in a variety of malignancy vaccines7,8and immunotherapeutic strategies against chronic infections and autoimmune disease9,10. Strong inter-individual variations are typically observed in the immunological and medical reactions of individuals exposed to KLH8. Methoctramine hydrate In-depth information about the dynamics and phenotype of the KLH-specific immune response may help to optimize its medical use and provide biomarkers for selecting individuals that will benefit most from KLH-based interventions. We currently lack appropriate monitoring tools that allow a detailed study of the KLH-specific B cell response. So far, B cell reactions to KLH have primarily been evaluated by quantifying KLH-specific antibodies in serum11,12,13,14,15,16. Direct longitudinal analysis of KLH-specific B cells in peripheral blood could provide novel information on the magnitude and phenotype of the KLH-specific B cell response. A number of recent studies used fluorescently-labeled antigens to directly monitor vaccine-, disease- or allergen-induced antigen-specific B cells17,18,19,20. In this study, we founded a novel flow-cytometric assay to detect, phenotype TEK and isolate KLH-specific B cells in peripheral blood inside a sensitive and specific manner. As proof of concept, we applied our novel assay to monitor KLH-specific B cell reactions inside a cohort of malignancy individuals that were vaccinated with autologous monocyte-derived matured dendritic cells (DC) loaded with KLH and tumor antigen. We found that the serum concentration of KLH-specific antibodies was highly correlated to the number and phenotype of KLH-specific B cells. Flow-cytometric isolation of the fluorescently labeled KLH-specific B cells allowedex vivoproduction of KLH-specific antibodies and confirmed the high specificity of the assay. By analyzing B cell maturation, we were able to visualize the dynamics of KLH-specific B cells following main as well as booster vaccination. Our novel assay allows detailed cellular monitoring of the KLH-specific B cell response. Applying this technique to the field of KLH-based interventions could provide new insight into the origin, development and maintenance of the KLH-specific response and may facilitate the development of novel KLH-applications. == Results == To gain an understanding of the B cell response to KLH, we set out to examine the rate of recurrence and phenotype of KLH-specific B cells across the DC vaccination course of 10 stage III melanoma individuals (Supplementary Table 1). To protect multiple phases of humoral immunity, we selected three time points during treatment to measure the main response as well as the recall response within each individual. To examine the primary response, baseline frequencies were determined 722 days before vaccination and after injection number 24 of the 1stcycle (designated 1stcycle). Recall reactions were identified after 3 injections of the 3rdvaccination cycle (designated 3rdcycle). == Detection of KLH-specific B cells via circulation cytometry is definitely specific and sensitive == First, we wanted to verify the specificity and level of sensitivity of the circulation cytometry-based detection assay here offered. For this purpose, PBMC samples were stained for common leukocyte markers, together with two preparations of fluorescently-labeled KLH using either FITC or ReadiLink 700/713. KLH-specific B cells were defined as double positive Methoctramine hydrate cells (KLH++) within the CD19+CD3CD14CD16CD56population (Fig..