Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV

Our previous investigation of a patient (pt1) with non-X-linked hyper-immunoglobulin M syndrome revealed a CD40-mediated defect in B cell activation that resulted in low CD23 expression and absence of germ-line transcription and class-switch recombination. These deficiencies were complemented in vitro by a high threshold of sustained signaling through CD40. To further analyze the signaling defect in pt1 B cells, two types of Epstein-Barr virus lymphoblastoid cell lines (LCLs) were generated that either constitutively expressed the viral transforming protein latent membrane protein-1 (LMP1; pt1-LCL) or expressed it under the control of a tet-inducible promoter (pt1-LCL^tet). Because LMP1 signals through the CD40 pathway, the pt1-LCL and pt1-LCL^tet lines allow comparison of downstream functions in response to either constitutive LMP1 signals or regulated LMP1 and CD40 signals. Immortalized pt1-LCLs were initially CD23^lo/CD38 ^hi and reverted to a CD23^hi/CD38^lo phenotype upon extended growth in culture, suggesting that the CD40 defect was reversed by selection and/or constitutive expression of LMP1. In contrast, pt1-LCL ^tet cells retained the CD23^lo/CD38^hi phenotype after extended periods of culture and failed to up-regulate CD23 in response to CD40 signals. Analysis of pt1-LCL^tet cells in response to the CD40 signals in the presence or absence of LMP1 revealed that mitogenic activation resulted only from LMP1 and not CD40, indicating a difference in the response of pt1 B cells to these two distinct signals. Together, these data demonstrate that the pt1-LCL^tet cells maintain the CD40-related defect and provide a unique approach to study the independent effects of LMP1- and CD40-directed signals.