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closeReferee comments: Referee 1
Posted by PLOS_ONE_Group on 25 Jan 2008 at 13:32 GMT
Referee 1's review:
The focus of this manuscript is a refinement of the previously-published technique of keeping human B cells alive for prolonged periods by repetitive stimulation via CD40 and the receptor for IL-4. The abstract is misleading, as it suggests that this has not been done before. In fact, this approach was published in 1991 in a Science paper by Jacques Banchereau and colleagues. The authors cite the paper in their manuscript, but seem to ignore it in the way they initially present the work as "the first" to enhance normal B cell survival in this way. This is not correct. Through manipulation of initial cell seeding number, they report longer lifespan for some of the cell lines, but that is the only real difference between the reports.
The are correct that this could be a useful alternative to the approach of EBV transformation, but this point was also made by the earlier study. Importantly, the chronically activated status of these cells (quite similar to EBV-transformed B cells) strongly limits their utility. It is also unknown whether cells that are chronically activated like this could be used for B cell vaccination or not. The stimuli through CD40 and the IL-4 receptor have many effects on B cells. They become highly activated, with a large number of phenotypic changes that have been well-documented in the published literature. No attempt was made here to assess whether in fact cells maintained in this way are effective at presenting antigen to, and stimulating, antigen-specific T cells in vitro or, most importantly, in vivo (necessary traits for B cells to be effective in vaccination). Did these B cells produce antibody? Show isotype switching? Accumulate somatic mutations in their Ig genes? It is notable on p. 9 that almost a third had an abnormal karyotype.
To be a true advance over the earlier study, the authors would need to show that these B cells can be useful in some line of inquiry. They cannot be used to study activation of human B cells, because, just like EBV-transformed B cells, they are already quite activated by the signals that maintain their survival. Some concrete demonstration of enhanced utility compared to EBV-transformed B cells would considerably improve what is currently a minimal increment of new information.
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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.
Author response to Referee 1
Moosmann1 replied to PLOS_ONE_Group on 29 Jan 2008 at 18:42 GMT
The above review referred to the first, considerably shorter version of our manuscript. The published version additionally contains Figures 6, 8, 9 and 10, related material in the Results and Discussion sections, and additional information requested by the referees.
However, our central claim has remained the same. It is this: we describe, for the first time, the conditional immortalization of normal human B cells by external stimulation.
Strikingly, referee 1 never discussed this claim. "Immortalization" was not mentioned in his/her review. Still, the referee came to the conclusion that our paper contains only a "minimal increment of new information". In our opinion, such a harsh conclusion cannot be justified as long as any discussion of our central claim is denied.
We summarize our response to further points of criticism made by referee 1:
1. In our opinion, we properly credited the seminal study by Banchereau et al. (our reference #18). Banchereau et al. described "long term human B cell lines" which, however, "were found to die out after 10 weeks for presently unexplained reasons." This culture period is far too short to consider whether these B cells might have been immortalized. Other investigators cultured CD40 B cells for similarly short periods. Therefore, the question whether CD40-stimulated B cells might be conditionally immortalized has not been raised by these investigators, and we believe we are the first to ask and answer this specific question.
2. In our opinion, CD40 B cells are not plainly "quite activated", but they are activated with respect to certain specific functions, and remain inactive with respect to other functions. Therefore, CD40 B cells can be used to study specific aspects of B cell activation. We give an example by showing that CD40 B cells can be stimulated by CpG oligonucleotides to produce certain cytokines (Figure 8).
3. It was not our intention to propose the use of CD40 B cells in vaccination, but as in vitro tools to expand specific T cells for therapy. Therefore, the referee's critical remarks on vaccination are valuable but do not apply to our study.
4. We complied with the referee's request to show that CD40 B cells are "useful". In our revised manuscript, we give examples showing that CD40 B cells can serve as a tool to study B cell activation (Figure 8) and stimulate antigen-specific T cells (Figure 9). However, the central message of this paper is not that conditionally immortalized CD40 B cells are useful, but that they exist. We intend to explore the usefulness of CD40 B cells in greater detail in future studies.