Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-seq data, as input and orders cells according to their developmental progression by pinpointing bifurcation points and labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points.

Kidney transplantation has been one of the major medical advances of the past 30 years. However, tissue availability remains a major obstacle. This can potentially be overcome by the use of undifferentiated or partially developed kidney precursor cells derived from early embryos and fetal tissue. Here, transplantation in mice reveals the earliest gestational time point at which kidney precursor cells, of both human and pig origin, differentiate into functional nephrons and not into other, non-renal professional cell types. Moreover, successful organogenesis is achieved when using the early kidney precursors, but not later-gestation kidneys. The formed, miniature kidneys are functional as evidenced by the dilute urine they produce. In addition, decreased immunogenicity of the transplants of early human and pig kidney precursors compared with adult kidney transplants is demonstrated in vivo. Our data pinpoint a window of human and pig kidney organogenesis that may be optimal for transplantation in humans.

The T-cell receptor (TCR) repertoire is formed by random recombinations of genomic precursor elements; the resulting combinatorial diversity renders unlikely extensive TCR sharing between individuals. Here, we studied CDR3b amino acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3 sequences, relative to private sequences, are two orders of magnitude more abundant on average, express restricted V/J segments, and feature high convergent nucleic acid recombination. Functionally, public sequences are enriched for MHC-diverse CDR3 sequences that were previously associated with autoimmune, allograft, and tumor-related reactions, but not with anti-pathogen-related reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are associated with different, MHC-dependent, V genes. Thus, despite their random generation process, TCR repertoires express a degree of uniformity in their post-genomic organization. These results, together with numerical simulations of TCR genomic rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing selection to generate a restricted subset of self-associated, public CDR3 TCR sequences, and invite reexamination of the basic mechanisms of T-cell repertoire formation. [Supplemental material is available for this article.]The genome provides the raw material for the somatic generation of enormous T-cell receptor (TCR) diversity. These receptors are generated through a random process of DNA rearrangement, which involves the recombination of the germline V, D, and J gene segments and the deletion and insertion of nucleotides at the V(D)J junctions. The variety of the resulting TCR repertoire is required to recognize a large and unpredictable range of antigens of foreign and self origin. The potential diversity of TCR molecules synthesized during the maturation of T cells in the thymus is estimated to be >10 15 for the mouse TCRA and TCRB repertoire (Casrouge et al. 2000) and >10 10 for the b segment of the TCR. In

Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase-labeled MSCs (Luc 1 MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune-deficient Balb-Nude or non-obese diabetic severe combined immunodeficiency (NOD-SCID) recipients. Graft rejection in re-challenge experiments infusing Luc 1 Fib into mice, which had previously rejected Luc 1 MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T-cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re-challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8 1 or TEA CD4 1 T cells exhibited a memory phenotype (CD122 1 , CD44 1 , and CD62L low ). Collectively, these results demonstrate that MSCs are not intrinsically immune-privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (''off the shelf'') MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long-term survival of allogeneic MSCs likely represents a major challenge. STEM CELLS

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity.DOI: http://dx.doi.org/10.7554/eLife.22057.001