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Affinity maturation of antibody responses depends on somatic hypermutation of the immunoglobulin V genes. Hypermutation is initiated specifically in proliferating B cells in lymphoid germinal centres but the signals driving this process remain unknown. This study identifies signals that promote V gene mutation in human germinal centre (GC) B cells in vitro. Single GC B cells were cultured by limiting dilution to allow detection of mutations arising during proliferation in vitro. Cells were first cultured in the presence of CD32L cell transfectants and CD40 anti

Background: Type 2 T-helper cells (Th2) are involved in the regulation of the humoral immune response against antigens and allergens and directly affect which isotype will be produced. The mechanism that regulates antigen-specific IgE secretion and immune deviation is still not known. Objectives: To delineate mechanisms behind antigen-specific IgE secretion we have used in vitro immunization and focused on T-cell phenotype and the activation status of the transcription factor NFkB. Methods: Peripheral blood lymphocytes (PBMC) from seronegative donors were immunized in vitro with a peptide consisting of both a T-cell and a B-cell epitope. Results Antigen-specific IgE antibodies could be detected after a primary immunization, during which T-helper cells secreted type 2 cytokines. Specific IgE was also detected in the secondary immunization, but due to a rapid polarization from Th2 to Th1 phenotype, exogenous IL-4 was required for the specific IgE secretion. Analysis of NFkB activation in B and T cells during primary and secondary immunization showed that NFkB could be detected in both B and T cells during primary immunization, but was dependent on exogenous IL-4 in the secondary immunization. Conclusion: This is the first evidence of antigen-specific IgE induction in vitro using naive B cells, demonstrating the involvement of T-helper cell phenotype and NFkB and demonstrates the usefulness of in vitro cultures to study the effect of antigens on human immunocytes.

Background: Type 2 T-helper cells (Th2) are involved in the regulation of the humoral immune response against antigens and allergens and directly affect which isotype will be produced. The mechanism that regulates antigen-specific IgE secretion and immune deviation is still not known. Objectives: To delineate mechanisms behind antigen-specific IgE secretion we have used in vitro immunization and focused on T-cell phenotype and the activation status of the transcription factor NFkB. Methods: Peripheral blood lymphocytes (PBMC) from seronegative donors were immunized in vitro with a peptide consisting of both a T-cell and a B-cell epitope. Results Antigen-specific IgE antibodies could be detected after a primary immunization, during which T-helper cells secreted type 2 cytokines. Specific IgE was also detected in the secondary immunization, but due to a rapid polarization from Th2 to Th1 phenotype, exogenous IL-4 was required for the specific IgE secretion. Analysis of NFkB activation in B and T cells during primary and secondary immunization showed that NFkB could be detected in both B and T cells during primary immunization, but was dependent on exogenous IL-4 in the secondary immunization. Conclusion: This is the first evidence of antigen-specific IgE induction in vitro using naive B cells, demonstrating the involvement of T-helper cell phenotype and NFkB and demonstrates the usefulness of in vitro cultures to study the effect of antigens on human immunocytes.