Abstract
BackgroundDifferentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells.ResultsWe perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing.ConclusionThe link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0957-5) contains supplementary material, which is available to authorized users.
Highlights
Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood
Single-cell RNA-seq links CD4+ T-cell division rates to differentiation state in an in vivo Th1 infection model To verify the link between cell cycle speed and differentiation rate in vivo and to ask if the model can be extended from Th2 to Th1 differentiation, we studied the CD4+ T-cell response against Plasmodium chabaudi AS (PcAS)
Amongst the ex vivo cycling cells, we confirm a correlation between cell cycle speed and differentiation. This finding supports a new concept during T helper (Th) cell primary activation: is cell cycle entry a sine qua non for differentiation but a greater level of differentiation is associated with faster cycling cells— in Th2 cells but universally during primary activation of different subtypes of T lymphocytes
Summary
Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. By sorting out separate cell populations from a single cell culture of asynchronized, dividing cells, we aimed to reduce the biological variability in cytokine exposure, confluence, etc With this approach, we minimize the biological noise in our data and focus entirely on the processes of cell division and differentiation. By counting cells in each cell generation using flow cytometry, we modelled the rates of death, division and differentiation using a discrete time Markov branching process. This revealed a higher cell division rate for differentiated cells compared with proliferating, activated cells. We validate those finding by DNA staining and by single-cell live imaging of Th2 cells. These in vitro data supported the idea of a fine-tuned relationship between cell cycle speed and differentiation status in CD4+ T cells
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