Referee 2's review:

Wiesner and colleagues investigated B cell immortalization through constant CD40L/Il-4 stimulation of primary human B cells from various starting amounts of PBMCs. Impressively the authors were able to establish significantly long term cultures from healthy human primary B cells, some continuing for >900 days without senescence and with maintenance of telomere length and telomerase activity. Overall a good paper that extends the analysis of B cell stimulation and establishes the technical aspects for generating B cell lines from primary sources possibly without viral transformation.

In critique, the authors found that the best cultures came from smaller starting amounts of PBMCs, with cell numbers in the range of 104-105 as presented in figure 1. In cultures starting from higher amounts (>106), B cells proliferation was decreased, and there was a high contamination rate of T cells. The authors contend that these are cytotoxic T cells due to the rapid destruction of the CD40L
L cells; this was not shown though. As a point of question, it was not completely clear from both the results and the methods/figure legends if the density of the
CD40L fibroblast and plating volume/surface area of the tissue culture well was increased to accommodate the higher volume of cells, because the reason for the difference between starting amounts of PBMCs and culture efficiency could have been access to proper stimulation from the CD40L feeder layer. In the methods, the authors discussed using a range of feeder layer cells in a 12 or 96 well format but were not detailed about the specifics for each experiment, these details would be valuable. It would be interesting (but not critical) as well to see if different types of CD40L such as commercial soluble forms could produce the same results. Also, it would be interesting to establish if the cultures were from an outgrowth of a single or small number of B cell clones or is the line polyclonal? An important follow-up to this story (as noted in the discussion) would be to test the efficiency of different B cell subsets to transform, possibly thru sorting/enriching the subsets. It would be important to know if a particular B cell subset is better at establishing the long term culture as PBMCs include a smorgasbord of B cell subsets. Finally, it would be interesting to note the immunoglobulin profile of the established cultures (specifically IgM/IgA/IgG). To avoid EBV, the authors should have used an EBV negative blood donor, that are understandably rare, but not hard to find and there is a simple serological test.
Further, it would be helpful if the authors showed the B cell lines that express EBV as a reference point in the PCR of figure 4. These lines were mentioned in the text but not shown in the PCR.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.