title: Single-cell analysis of CD4+ T-cell differentiation reveals three major cell states and progressive acceleration of proliferation.
abstract: BACKGROUND
  Differentiation 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.|Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Valentina
authors.initials: V
authors.lastname: Proserpio
authors.affiliation: Department of Physics, University of Naples Federico II, CNR-Spin, Istituto Nazionale di Fisica Nucleare (INFN), Napoli, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Piccolo
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.|Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Liora
authors.initials: L
authors.lastname: Haim-Vilmovsky
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.
authors.forename: Gozde
authors.initials: G
authors.lastname: Kar
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.|Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Tapio
authors.initials: T
authors.lastname: Lönnberg
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.
authors.forename: Valentine
authors.initials: V
authors.lastname: Svensson
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.|Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Jhuma
authors.initials: J
authors.lastname: Pramanik
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.|Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Kedar Nath
authors.initials: KN
authors.lastname: Natarajan
authors.affiliation: Cavendish Laboratory, University of Cambridge, Madingley Road, Cambridge, CB3 0HE, UK.
authors.forename: Weichao
authors.initials: W
authors.lastname: Zhai
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK.
authors.forename: Xiuwei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Centre for Stem Cells and Regenerative Medicine, Kings College London, London, SE1 9RT, UK.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Donati
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK.
authors.forename: Melis
authors.initials: M
authors.lastname: Kayikci
authors.affiliation: Cavendish Laboratory, University of Cambridge, Madingley Road, Cambridge, CB3 0HE, UK.
authors.forename: Jurij
authors.initials: J
authors.lastname: Kotar
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK.
authors.forename: Andrew N J
authors.initials: AN
authors.lastname: McKenzie
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Ruddy
authors.initials: R
authors.lastname: Montandon
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Oliver
authors.initials: O
authors.lastname: Billker
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.|Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Woodhouse
authors.affiliation: Cavendish Laboratory, University of Cambridge, Madingley Road, Cambridge, CB3 0HE, UK.
authors.forename: Pietro
authors.initials: P
authors.lastname: Cicuta
authors.affiliation: Department of Physics, University of Naples Federico II, CNR-Spin, Istituto Nazionale di Fisica Nucleare (INFN), Napoli, Italy. mario.nicodemi@na.infn.it.
authors.forename: Mario
authors.initials: M
authors.lastname: Nicodemi
authors.affiliation: EMBL, European Bioinformatics Institute (EBI), Hinxton, CB10 1SD, UK. st9@sanger.ac.uk.|Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. st9@sanger.ac.uk.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Teichmann
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D008288:Malaria / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D059467:Transcriptome
chemical_list: D016207:Cytokines
keywords: Adaptive immunity; CD4+ T cells; Cell cycle; Differentiation; Live imaging; Single-cell RNA-seq