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Supplementary Figure 1 Modelling of the proliferation and death of SCID B cells. The numbers of SCID () and RAG1-/- () B cells present in each division cohort of (A) non-switching cultures and (B) switching cultures were estimated using bead counts and the CFSE profiles shown in Fig 6B. The frequencies of cells undergoing switching in each division number were estimated as shown in Fig 6C. Predicted distributions of SCID B cells across divisions were calculated using this data under two conditions: (short dash), a constant frequency of SCID B cells died in each cell division in excess to the death of control RAG-/- B cells; (long dash), in addition, the SCID cells that failed to successfully switch in each division (in comparison to RAG1-/- B cells) also died. Methods By ignoring the cell death that occurred in the cultures of RAG1-/- B cells, the number of founder cells (RAGN0n) producing the viable cells (RAGNPn) observed in each division cohort, n, could be calculated from the equation RAGN0n=RAGNPn/2n. In this way, RAGN0n was calculated for the division cohorts (n = 0 to 8) present in the RAG1-/- B cell cultures. Matrices were then produced in which these founder cells were tracked as they progressed through cell doublings with the addition of a theoretical constant fraction of dying cells, CD, where CD ranged from 0 to 1. Thus, with a constant excess death in SCID B cells over RAG1-/- B cells of 10% per cell division (CD=0.1), the number of viable SCID cells that would be detected in each division cohort at the end of culture is given by: SCIDNPo=RAGN00x0.91x20; SCIDNP1=RAGN01x0.92x21; and so on (ie: SCIDNPn=RAGN0nx(1-CD)n+1x2n). Such modelling was carried out with increasing values of CD using input data from the RAG1-/- B cell cultures, until the sum of SCIDNPn across all divisions was equal to the total number of viable cells actually found in our SCID B cell cultures. Additional modelling involved incorporating the death of all cells that failed to switch successfully in the SCID B cell cultures relative to the RAG1-/- B cell cultures. This was done by calculating the difference between SCID and RAG1-/- B cells in switching frequency (SDn = 0 to 1) for each serial division, n, then “killing” those cells that failed to switch in the SCID B cell cultures when performing the matrix analysis (ie: SCID NPn=RAGN0nx(1-SDn)x(1-CD)n+1x2n). Discussion of Supplementary Figure 1 We modelled the death of CFSE-labelled SWHELSCID cells using an approach similar to Gett et al. (2000) and Hasbold, et al. (1999) (see Methods above). Using this approach to analyse non-switching (CD40L + anti-CD38 + anti-CD80) cultures of SCID and RAG1-/- B cells, we could show that a constant excess death rate in SCID B cells over RAG1-/- B cells of 11.0% per cell division (CD=0.11) very accurately predicted the total number of viable SCID B cells that in fact remained at the end of culture as well as their distribution across serial cell divisions (Suppl Fig 1A). These results were consistent with the conclusion that the SCID mutation increases cell death in proliferating B cells independently of switching. A comparable CD value of 16.1% predicted the total number of viable SCID B cells obtained in switching (CD40L + IL4) cultures, but did not predict the observed distribution of SCID B cells across serial cell divisions (Suppl Fig 1). In other words, the modelling strategy revealed that the difference in cell death between SCID and RAG1-/- B cells in CD40L + IL4 cultures was not constant through serial divisions, and was in fact greater in cells that were slower to divide. This was true also of LPS + TGF cultures (data not shown). Since the death of SCID cells which failed to complete switching would skew the distribution of cells even further towards earlier division numbers (see Suppl Fig 1B), the discrepancy between modelled and observed SCID cell numbers cannot be ascribed to death as a consequence of failed switching. We were thus unable to determine whether or not failure to switch in SCID B cells is lethal, because we have so far been unable to identify the cause(s) of the differences in viability and proliferation observed between switching SCID and RAG1-/- B cell cultures. References Gett, A.V. and Hodgkin, P.D. (2000) A cellular calculus for signal integration by T cells. Nat. Immunol., 1, 239-244. Hasbold, J., Gett, A.V., Rush, J.S., Deenick, E., Avery, D., Jun, J. and Hodgkin, P.D. (1999) Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester. Immunol. Cell Biol., 77, 516-522.