More diversity at the top: A detailed knowledge of cell differentiation hierarchies is important for understanding diverse biological processes such as organ development, tissue regeneration, and cancer. Single-cell RNA sequencing can help elucidate these hierarchies, but it requires reliable computational methods for predicting cell lineage trajectories. Gulati et al. developed CytoTRACE, a computational framework based on the simple observation that transcriptional diversity—the number of genes expressed in a cell—decreases during differentiation. CytoTRACE outperformed other methods in several test cases and was successfully applied to study cellular hierarchies in healthy and tumor tissue. Science , this issue p. 405

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells.CD47mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Kwakkenbos et al. describe an in vitro method to generate antibody-secreting B cell lines from human peripheral blood memory B cells by transducing them with retroviral vectors encoding Bcl-6 and Bcl-xL. The approach can be used to stably and simultaneously produce high levels of B cell receptor (BCR) on the cell surface and secreted immunoglobulins, useful for studying BCR signaling and producing antigen-specific antibodies. The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)–positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.

Background & Aims Paneth cells contribute to the small intestinal niche of Lgr5⁺ stem cells. Although the colon also contains Lgr5⁺ stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5⁺ stem cells. Methods We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117⁺ crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit⁺ goblet cells were interdigitated with Lgr5⁺ stem cells. In vivo, this colonic cKit⁺ population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit⁺ cells. When isolated from mouse colon, cKit⁺ cells promoted formation of organoids from Lgr5⁺ stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit⁺ cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5⁺ stem cells.