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T-cell stimuli independently sum to regulate an inherited clonal division fate

View Article: PubMed Central - PubMed

ABSTRACT

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities.

No MeSH data available.


Inter-clonal variation in DD is regulated by receptor sensitivity and clonal experience.(a–d) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were sorted into (a) CD28 high and low expressing populations and stimulated with N4 peptide±αCD28 agonist antibody (20 μg ml−1). All cultures contained S4B6 (25 μg ml−1). Cell number versus (b) time and (c) mean division number (MDN) were measured. (d) An estimation of the percentage of the starting cells whose progeny are contributing to the response at that time point, calculated by removing the effect of cell expansion (percentage cohort number, see Methods) versus MDN. (e–h) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were stimulated with N4 peptide and αCD28 (2 μg ml−1) for 25 h then sorted for (e) IL-2Rα high or low expression. Cells were placed back into culture±hIL-2 (3.16 U ml−1) and cell number versus (f) time and (g) MDN were measured and (h) percentage cohort number versus MDN calculated. Arrows indicate the difference in mDD between populations when no additional ligand (grey) or ligand at the specified concentration (black) was added to the culture. Representative of two (a–d) or three (e–h) independent experiments. Mean±s.e.m. of triplicate culture wells.
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f6: Inter-clonal variation in DD is regulated by receptor sensitivity and clonal experience.(a–d) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were sorted into (a) CD28 high and low expressing populations and stimulated with N4 peptide±αCD28 agonist antibody (20 μg ml−1). All cultures contained S4B6 (25 μg ml−1). Cell number versus (b) time and (c) mean division number (MDN) were measured. (d) An estimation of the percentage of the starting cells whose progeny are contributing to the response at that time point, calculated by removing the effect of cell expansion (percentage cohort number, see Methods) versus MDN. (e–h) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were stimulated with N4 peptide and αCD28 (2 μg ml−1) for 25 h then sorted for (e) IL-2Rα high or low expression. Cells were placed back into culture±hIL-2 (3.16 U ml−1) and cell number versus (f) time and (g) MDN were measured and (h) percentage cohort number versus MDN calculated. Arrows indicate the difference in mDD between populations when no additional ligand (grey) or ligand at the specified concentration (black) was added to the culture. Representative of two (a–d) or three (e–h) independent experiments. Mean±s.e.m. of triplicate culture wells.

Mentions: To identify possible sources of individual founder cell variation we asked whether differences in critical receptor levels for the stimuli tested in Figs 2, 3, 4, 5 correlated with subsequent family clone size. CD28 and interleukin-2 receptor chain alpha (IL-2Rα) levels just prior to the first division were relatively uniform (Supplementary Fig. 8) (Spearman's correlation of 0.16), supporting the finding of independence of signal effect on DD in clonal families. To investigate if CD28 levels influenced DD, we sorted naive OT-I/Bcl2l11−/− CD8+ T cells into CD28 high (CD28hi) and low (CD28lo) expressing populations (Fig. 6a) and measured the effect on mDD. Residual αCD28 antibody from sorting had little effect on cell expansion (open circles, Fig. 6b–d). When αCD28 was added to the culture the CD28hi population had a ∼0.6 division increase in mDD relative to CD28lo cells (black arrow, Fig. 6c,d), resulting in an ∼50% increase in cell expansion (closed circles, Fig. 6b,c). Thus, differences in initial CD28 receptor level exhibited by the naive T-cell population did contribute to the founder cell variation in DD.


T-cell stimuli independently sum to regulate an inherited clonal division fate
Inter-clonal variation in DD is regulated by receptor sensitivity and clonal experience.(a–d) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were sorted into (a) CD28 high and low expressing populations and stimulated with N4 peptide±αCD28 agonist antibody (20 μg ml−1). All cultures contained S4B6 (25 μg ml−1). Cell number versus (b) time and (c) mean division number (MDN) were measured. (d) An estimation of the percentage of the starting cells whose progeny are contributing to the response at that time point, calculated by removing the effect of cell expansion (percentage cohort number, see Methods) versus MDN. (e–h) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were stimulated with N4 peptide and αCD28 (2 μg ml−1) for 25 h then sorted for (e) IL-2Rα high or low expression. Cells were placed back into culture±hIL-2 (3.16 U ml−1) and cell number versus (f) time and (g) MDN were measured and (h) percentage cohort number versus MDN calculated. Arrows indicate the difference in mDD between populations when no additional ligand (grey) or ligand at the specified concentration (black) was added to the culture. Representative of two (a–d) or three (e–h) independent experiments. Mean±s.e.m. of triplicate culture wells.
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f6: Inter-clonal variation in DD is regulated by receptor sensitivity and clonal experience.(a–d) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were sorted into (a) CD28 high and low expressing populations and stimulated with N4 peptide±αCD28 agonist antibody (20 μg ml−1). All cultures contained S4B6 (25 μg ml−1). Cell number versus (b) time and (c) mean division number (MDN) were measured. (d) An estimation of the percentage of the starting cells whose progeny are contributing to the response at that time point, calculated by removing the effect of cell expansion (percentage cohort number, see Methods) versus MDN. (e–h) Naive CTV-labelled OT-I/Bcl2l11−/− CD8+ T cells were stimulated with N4 peptide and αCD28 (2 μg ml−1) for 25 h then sorted for (e) IL-2Rα high or low expression. Cells were placed back into culture±hIL-2 (3.16 U ml−1) and cell number versus (f) time and (g) MDN were measured and (h) percentage cohort number versus MDN calculated. Arrows indicate the difference in mDD between populations when no additional ligand (grey) or ligand at the specified concentration (black) was added to the culture. Representative of two (a–d) or three (e–h) independent experiments. Mean±s.e.m. of triplicate culture wells.
Mentions: To identify possible sources of individual founder cell variation we asked whether differences in critical receptor levels for the stimuli tested in Figs 2, 3, 4, 5 correlated with subsequent family clone size. CD28 and interleukin-2 receptor chain alpha (IL-2Rα) levels just prior to the first division were relatively uniform (Supplementary Fig. 8) (Spearman's correlation of 0.16), supporting the finding of independence of signal effect on DD in clonal families. To investigate if CD28 levels influenced DD, we sorted naive OT-I/Bcl2l11−/− CD8+ T cells into CD28 high (CD28hi) and low (CD28lo) expressing populations (Fig. 6a) and measured the effect on mDD. Residual αCD28 antibody from sorting had little effect on cell expansion (open circles, Fig. 6b–d). When αCD28 was added to the culture the CD28hi population had a ∼0.6 division increase in mDD relative to CD28lo cells (black arrow, Fig. 6c,d), resulting in an ∼50% increase in cell expansion (closed circles, Fig. 6b,c). Thus, differences in initial CD28 receptor level exhibited by the naive T-cell population did contribute to the founder cell variation in DD.

View Article: PubMed Central - PubMed

ABSTRACT

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities.

No MeSH data available.