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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.