Differentiation of naïve CD4 + T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a * Correspondence to: st9@sanger.ac.uk, Ashraful.Haque@qimrberghofer.edu.au or stegle@ebi.ac.uk. # denotes equal contribution † denotes equal contribution Author contributions TL and KRJ performed the single-cell RNA-seq experiments. VS developed the GPfates model in collaboration with MZ, NDL, OS and SAT. DFR and WRH generated the PbTII mouse model. KRJ, RM, IS, MSFS, LGF, ASN, UL, FSFG, PTB and CRE performed the mouse experiments. TL, VS, KRJ, LHL and FOB analysed the data and interpreted the results MJTS performed the TCR clonality analysis. TL, KRJ, RM, OB, AH and SAT designed the experiments. OS, AH and SAT cosupervised the study. TL, VS, KRJ, OS, AH and SAT wrote the manuscript. All authors have read and approved the manuscript. Competing interestsThe authors declare no competing interests. Data and materials availabilityThe data presented in this paper is publically available in the ArrayExpress database with accession number E-MTAB-4388. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates.

Significance Immunoregulatory poperties have been principally ascribed to various mature immune cell types, including regulatory B cells. An immature B-cell progenitor population endowed with suppressive properties per se or after differentiation into more mature regulatory B cells has not been demonstrated as yet. We now describe a pro–B-cell progenitor population that emerged upon stimulation with the Toll-like receptor-9 ligand CpG and prevented disease upon adoptive transfer into autoimmune type 1 diabetes-prone mice. Effector T cells were the target of immunoregulatory pro-B cells and of their mature progeny. Such protective pro-B cells could be instrumental for cell therapy of autoimmune diseases.

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.

Mechanisms of protection against autoimmune diseases by transplantation of autologous hematopoietic progenitors remain poorly defined. We recently demonstrated that, unlike medullary hematopoietic stem cells (HSCs), mobilized hematopoietic progenitors (HPCs) stimulate peripheral Foxp3 ؉ regulatory T cell (Treg)-expansion through cell-contact activation of Notch signaling and through as yet undetermined soluble factor(s), distinct from TGF-␤1. Herein we identified one such soluble factor as granulocyte macrophage-colony stimulating factor (GM-CSF), which is produced at higher levels by HPCs than HSCs and whose neutralization significantly reduces the growthpromoting effect of HPCs on Treg. IntroductionMobilized peripheral blood stem cells are increasingly used as an alternative to bone marrow (BM) cells for allogeneic transplantation in cancer patients and, more recently, for autologous transplantation in patients with severe autoimmune diseases. Although in the allogeneic setting regulatory T cell (Treg) accumulation triggered by myeloid Gr1 ϩ CD11b ϩ suppressor cells (MSC) reduces acute graft-versus-host disease (GVHD) 1 but instead worsens chronic GVHD, 2 in autoimmune diseases the expansion of Treg is expected to be beneficial. In the experimental model of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice, we have recently demonstrated 3 that Lin Ϫ Sca1 hi ckit hi Flt3 ϩ CD34 ϩ CD106 ϩ CD127 Ϫ multipotent hematopoietic progenitor cells (HPCs) mobilized to the spleen by a combination of granulocytecolony stimulating factor (G-CSF) and FMS-like tyrosine kinase 3 ligand (Flt3L), have a tolerogenic potential and that their transplantation halts autoimmune diabetes. In contrast, nonmobilized Lin Ϫ Sca1 hi ckit hi Flt3 Ϫ CD34 Ϫ CD106 lo CD127 Ϫ medullary hematopoietic stem cells (HSCs) have no such effect. This difference was linked to the capacity of HPCs, but not HSCs, to drive the expansion of host-derived CD4 ϩ CD25 ϩ Foxp3 ϩ Treg. Furthermore, restoration of Treg numbers to normal values took place after transplantation with hematopoietic progenitors in patients with juvenile rheumatoid arthritis. 4 In view of all these data, the elucidation of the mechanisms underlying Treg expansion by hematopoietic progenitors became essential. We have demonstrated that a cell-to-cell interaction between HPCs and Treg triggers the stimulation of Notch signaling in Treg and their subsequent proliferation. However, separating HPCs from Treg 3 in transwell experiments only partially blocked Treg expansion by HPCs, suggesting that soluble factor(s) were implicated as well.We now report the identification of one such soluble factor as granulocyte macrophage-colony stimulating factor (GM-CSF), which is produced at higher levels by HPCs than HSCs, and promotes expansion of functional Treg through its specific ␣-chain receptor CD116. Methods MiceWild-type and Rag2 Ϫ/Ϫ -NOD mice were bred in our animal facility under specific pathogen-free conditions. Live animal experiments were approved by the Ministère de l'Agricul...