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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.


Related in: MedlinePlus

Clonal T-cell family proliferation is synchronized.OT-I/Bcl2l11−/− CD8+ T cells were processed, stimulated and analysed as described in Fig. 2. All cultures contained S4B6 (25 μg ml−1). (a) N4+αCD28+IL-2 stimulated population of control cells labelled with CTV and CFSE to distinguish generation number for four distinct labelling configurations. Cells were separated into CPD+ and CPD−, allowing division tracking in eight populations per well. (b) Examples of clonal progeny detected in individual wells. Example data shown for 72 h time point. (c) Generation number of progeny cells detected from individual clonal families at each time point from N4+αCD28+IL-2 stimulation condition. Progeny cells were classified as quiescent based upon small cell size (refer to Methods). Clonal range=maximum−minimum generation number. The symbol # at the end of a line denotes clones where all progeny cells were detected. Founder cell input after sorting was 80, 80 and 64 for data from 54, 62 and 72 h respectively. Results from a second independent experiment are shown in Supplementary Fig. 3.
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f3: Clonal T-cell family proliferation is synchronized.OT-I/Bcl2l11−/− CD8+ T cells were processed, stimulated and analysed as described in Fig. 2. All cultures contained S4B6 (25 μg ml−1). (a) N4+αCD28+IL-2 stimulated population of control cells labelled with CTV and CFSE to distinguish generation number for four distinct labelling configurations. Cells were separated into CPD+ and CPD−, allowing division tracking in eight populations per well. (b) Examples of clonal progeny detected in individual wells. Example data shown for 72 h time point. (c) Generation number of progeny cells detected from individual clonal families at each time point from N4+αCD28+IL-2 stimulation condition. Progeny cells were classified as quiescent based upon small cell size (refer to Methods). Clonal range=maximum−minimum generation number. The symbol # at the end of a line denotes clones where all progeny cells were detected. Founder cell input after sorting was 80, 80 and 64 for data from 54, 62 and 72 h respectively. Results from a second independent experiment are shown in Supplementary Fig. 3.

Mentions: Figure 3a–c and Supplementary Fig. 3 displays results of an application of the multiplex clonal stimulation assay to OT-I/Bcl2l11−/− CD8+ T cells stimulated by N4, αCD28 and IL-2 (added as human IL-2 (hIL-2) to overcome blocking by S4B6). Fig. 3a presents a population control culture where 500–2,000 cells were sorted from each of eight labelled groups, stimulated and harvested at 72 h post stimulation. As described in Fig. 2, the data in Fig. 3a enabled the identification of generation-gates for each dye-combination. Fig. 3b shows two single co-cultured wells harvested at the same time, with the population-determined gates overlaid, allowing the determination of the generation of each individual cell in each clonal family. These data illustrate how the multiplexing system permits distinct families to be isolated and followed in the co-culture, empowering analysis of familial concordance under these, and altered, culture conditions.


T-cell stimuli independently sum to regulate an inherited clonal division fate
Clonal T-cell family proliferation is synchronized.OT-I/Bcl2l11−/− CD8+ T cells were processed, stimulated and analysed as described in Fig. 2. All cultures contained S4B6 (25 μg ml−1). (a) N4+αCD28+IL-2 stimulated population of control cells labelled with CTV and CFSE to distinguish generation number for four distinct labelling configurations. Cells were separated into CPD+ and CPD−, allowing division tracking in eight populations per well. (b) Examples of clonal progeny detected in individual wells. Example data shown for 72 h time point. (c) Generation number of progeny cells detected from individual clonal families at each time point from N4+αCD28+IL-2 stimulation condition. Progeny cells were classified as quiescent based upon small cell size (refer to Methods). Clonal range=maximum−minimum generation number. The symbol # at the end of a line denotes clones where all progeny cells were detected. Founder cell input after sorting was 80, 80 and 64 for data from 54, 62 and 72 h respectively. Results from a second independent experiment are shown in Supplementary Fig. 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121331&req=5

f3: Clonal T-cell family proliferation is synchronized.OT-I/Bcl2l11−/− CD8+ T cells were processed, stimulated and analysed as described in Fig. 2. All cultures contained S4B6 (25 μg ml−1). (a) N4+αCD28+IL-2 stimulated population of control cells labelled with CTV and CFSE to distinguish generation number for four distinct labelling configurations. Cells were separated into CPD+ and CPD−, allowing division tracking in eight populations per well. (b) Examples of clonal progeny detected in individual wells. Example data shown for 72 h time point. (c) Generation number of progeny cells detected from individual clonal families at each time point from N4+αCD28+IL-2 stimulation condition. Progeny cells were classified as quiescent based upon small cell size (refer to Methods). Clonal range=maximum−minimum generation number. The symbol # at the end of a line denotes clones where all progeny cells were detected. Founder cell input after sorting was 80, 80 and 64 for data from 54, 62 and 72 h respectively. Results from a second independent experiment are shown in Supplementary Fig. 3.
Mentions: Figure 3a–c and Supplementary Fig. 3 displays results of an application of the multiplex clonal stimulation assay to OT-I/Bcl2l11−/− CD8+ T cells stimulated by N4, αCD28 and IL-2 (added as human IL-2 (hIL-2) to overcome blocking by S4B6). Fig. 3a presents a population control culture where 500–2,000 cells were sorted from each of eight labelled groups, stimulated and harvested at 72 h post stimulation. As described in Fig. 2, the data in Fig. 3a enabled the identification of generation-gates for each dye-combination. Fig. 3b shows two single co-cultured wells harvested at the same time, with the population-determined gates overlaid, allowing the determination of the generation of each individual cell in each clonal family. These data illustrate how the multiplexing system permits distinct families to be isolated and followed in the co-culture, empowering analysis of familial concordance under these, and altered, culture conditions.

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.


Related in: MedlinePlus