WNT-SHH Antagonism Specifies and Expands Stem Cells prior to Niche Formation.

Adult stem cell (SC) maintenance and differentiation are known to depend on signals received from the niche. Here, however, we demonstrate a mechanism for SC specification and regulation that is niche independent. Using immunofluorescence, live imaging, genetics, cell-cycle analyses, in utero lentiviral transduction, and lineage-tracing, we show that in developing hair buds, SCs are born from asymmetric divisions that differentially display WNT and SHH signaling. Displaced WNT(lo) suprabasal daughters become SCs that respond to paracrine SHH and symmetrically expand. By contrast, basal daughters remain WNT(hi). They express but do not respond to SHH and hence maintain slow-cycling, asymmetric divisions. Over time, they become short-lived progenitors, generating differentiating daughters rather than SCs. Thus, in contrast to an established niche that harbors a fixed SC pool whose expelled progeny differentiate, asymmetric divisions first specify and displace early SCs into an environment conducive to expansion and later restrict their numbers by switching asymmetric fates.

CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours.

Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.

Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer.

Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets.

Progenitors oppositely polarize WNT activators and inhibitors to orchestrate tissue development.

To spatially co-exist and differentially specify fates within developing tissues, morphogenetic cues must be correctly positioned and interpreted. Here, we investigate mouse hair follicle development to understand how morphogens operate within closely spaced, fate-diverging progenitors. Coupling transcriptomics with genetics, we show that emerging hair progenitors produce both WNTs and WNT inhibitors. Surprisingly, however, instead of generating a negative feedback loop, the signals oppositely polarize, establishing sharp boundaries and consequently a short-range morphogen gradient that we show is essential for three-dimensional pattern formation. By establishing a morphogen gradient at the cellular level, signals become constrained. The progenitor preserves its WNT signaling identity and maintains WNT signaling with underlying mesenchymal neighbors, while its overlying epithelial cells become WNT-restricted. The outcome guarantees emergence of adjacent distinct cell types to pattern the tissue.

A large peptidome dataset improves HLA class I epitope prediction across most of the human population.

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

Activin A regulates porcine follicle-stimulating hormone beta-subunit transcription via cooperative actions of SMADs and FOXL2.

Activins stimulate FSH synthesis and secretion by pituitary gonadotrope cells. Activin A induction of porcine and murine FSHß (Fshb) gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor L2 (FOXL2). Using both heterologous and homologous cell models, we demonstrate that FOXL2 functionally synergizes with SMAD3/4 to stimulate porcine Fshb promoter-reporter activity. We further show that endogenous FOXL2 and SMAD2/3 physically interact in homologous cells. We identify two composite cis-elements of adjacent FOXL2 and SMAD binding sites in the proximal porcine Fshb promoter that mediate activin A, FOXL2, and SMAD3 actions. FOXL2 can bind these elements independently of SMADs, whereas SMAD3/4 binding requires high-affinity FOXL2 binding. Conversely, FOXL2 alone is insufficient to regulate Fshb transcription and requires SMADs to induce promoter activity. Collectively, our data suggest a model in which activins stimulate formation and nuclear accumulation of SMAD3/4 complexes, which interact with FOXL2 bound to at least two proximal promoter elements. This association stabilizes SMAD3/4 binding to adjacent SMAD binding elements. SMAD-FOXL2 complexes then mediate activation of transcription through a currently unknown mechanism. Conservation of one of the two composite cis-elements suggests that this may form part of a general mechanism whereby activins regulate Fshb subunit transcription and FSH synthesis.