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Investigation of the molecular processes which control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte associated pathogenesis. Adenovirus-mediated gene transfer provided a powerful tool to investigate these processes. We have previously demonstrated that adenoviral vector Ad5/F35 transduces plasma cell lines at a higher efficiency than primary B cells, owing to differences in intracellular trafficking. Given that phosphatases are effectors of intracellular trafficking, here we have analyzed the effects of a panel of phosphatase inhibitors on Ad5/F35 transduction efficiency in B lymphocytes in the present study. FACS analysis was conducted to determine Ad5/F35-EYFP transduction efficiency in lymphoid cells, including human primary B cells, following serine/threonine phosphatase (PSP) inhibitor treatment. We further used confocal microscopy to analyze intracellular trafficking and fate of CY3 labeled Ad5/F35 vectors, in PSP treated lymphoid cell. Finally, we analyzed the MAPK pathway by Western blot in PSP treated cells. Adenoviral transduction efficiency was unresponsive to inhibition of PP1 whereas inhibition of PP2A by cantharidic acid, or PP1 and PP2A by okadaic acid, substantially increased transduction efficiency. Importantly, confocal microscopy analyses revealed that inhibition of PP2A shut down adenovirus recycling. Moreover, inhibition of PP2A resulted in increased phosphorylation of AKT, ERK1/2 and MEK1/2. Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells following PP2A inhibition. Our results are in agreement with reports indicating that PP2A is involved in the formation of recycling vesicles and might be of interest for gene therapy applications.

2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

Adenoviral gene transfer into human B lymphocytes and haematopoietic progenitors would allow the characterization of their function on cellular growth, differentiation and survival. Efficient gene expression is however strongly dependent on the promoter used. In this study, we investigated the relative strength of various promoters by following and measuring the expression of the reporter gene EYFP in human peripheral B lymphocytes, cord blood CD34(+) cells and the megakaryocytic cell line M-07e. The murine PGK promoter provided the best level of transgene expression in CD34(+) cells among the four promoters tested, followed closely by the CMV promoter, and to a lesser extend by a CMV promoter with a beta-globin/IgG chimeric intron, whereas the human CD40 promoter provided the lowest levels of expression. In contrast, the strongest promoters in B lymphocytes were the two CMV promoters. Surprisingly, even the best promoters were unable to induce transgene expression in more than 75-80% of the primary B and CD34(+) cells, even though 100% of the cells were infected. Finally and in contrast to retroviruses, only a minority of B lymphocytes and CD34(+) cells were able to induce the transcription of IRES-containing bicistronic expression cassettes from adenovirus.

The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34 + progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40–CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than naive B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.