HUMAN B CELL DIFFERENTIATION IN A MODEL SYSTEM

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The overall goal of this is to understand the regulation of immunoglobulin class switch recombination in human B cells. Specifically, we will use a model system that is comprised of a human B cell lymphoma cell line, RAMOS 266, that has the capacity to undergo isotype differentiation in response to cytokine and T cell-mediated signals. In addition, it was found that initial activation of the RAMOS B cells could be measured by up-regulation of surface CD23 and was dependent on the CD40 ligand (CD40-L) expressed on the surface of the D1.1 T cells. We will take advantage of RAMOS 266's capacity to undergo differentiation in culture to assess the transcriptional activation of the different constant region genes in response to cytokines and T cell help. In this project, T cell help will be defined as signaling through the CD40-L. Initial experiments will be carried out using RT-PCR on RAMOS mRNA isolated after exposure to CD40-L and/or cytokines. We will assess the response of the different isotype classes and subclasses to these signals and evaluate the response in terms of establishing whether class switching is proceeding by a directed or stochastic process. Our analyses will include measuring the germline (I- CH) transcript expression as well as the expression of mature (VDJ-CH) transcripts. To study more completely the regulation of isotype switching, we will analyze switch variants of RAMOS 266 cells and determine their capacity for further switch events. Additionally, we plan to evaluate the rearrangement events and the transcriptional response on the non- productive alleles of the switch variants to further establish a mechanism for the regulation of class switch. And finally, we will mutate the I gamma I exon of the heavy chain C gamma 1 region using homologous recombination. We will identify the rearrangement status of the non- productive allele after simulation with cytokines and T cell contact.