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.
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.
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 WNTlo suprabasal daughters become SCs that respond to paracrine SHH and symmetrically expand. By contrast, basal daughters remain WNThi. 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.
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•Asymmetric divisions within hair buds differentially partition WNT and SHH signaling•This partitioning couples asymmetric cell division to asymmetric cell fate•WNTlo daughters that respond to, but do not produce, SHH expand symmetrically•WNThi SHH-producing cells ultimately cease producing stem cell daughters
A mechanism for specifying stem cell numbers and identity that is independent of a physical niche operates during skin development: reciprocal signaling between an asymmetrically dividing and transient population of cells and their fast-cycling, symmetrically dividing sisters fuels the regulatory system.
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.
BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid ...tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).
Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies
, 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 efficacy
. 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.
Abstract
Though CAR T cell therapy has had success treating hematologic malignancies, these treatments have shown only modest efficacy in solid tumors. Little is known about the necessary molecular ...components required for CAR T cell cytotoxicity, especially in the context of solid tumors. We investigated the role of IFNgR signaling on solid tumors and discovered across tumor types (including glioblastoma, pancreatic, ovarian and lung) loss of IFNgR1 signaling resulted lower CAR T cell cytotoxicity in vitro and that this was irrespective of target antigen (including endogenous EGFR, IL13Ra2, mesothelin, and exogenous CD19). We also validated this in orthotopic xenograft models of glioblastoma and pancreatic cancer in vivo. CAR T cells exposed to wildtype (WT) or IFNgR1 KO tumor had similar transcriptional profiles, but the tumor cells had different signatures in response to CAR T cell co-culture. Further investigation showed marked upregulation of cell adhesion pathways in WT cells compared to IFNgR1 KO. We utilized microscopy with acoustic force and discovered CAR T cells had lower cell binding avidity to cells lacking IFNgR1 compared to WT cells. Following CAR T cell exposure, Intercellular Adhesion Molecule 1 (ICAM-1) was strongly upregulated at both the transcriptional and protein levels in WT cells but not IFNgR1 KO cells. We overexpressed ICAM-1 on IFNgR1 KO tumor cells and observed that CAR T cell binding avidity and cytotoxicity was restored to that of WT levels. This work highlights the importance of CAR T cell binding avidity and adhesion for optimal cytotoxicity. Better understanding of CAR T cell function will inform future construct design for successful therapies against solid tumors.
RCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.
Activins stimulate FSH synthesis and secretion by pituitary gonadotrope cells. Activin A induction of porcine and murine FSHβ (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.
SMAD proteins and FOXL2 physically and functionally interact to mediate activin-induced FSHβ subunit transcription in gonadotrope cells.
TGF-β signalling regulates various cellular activities throughout development and adulthood. Disruptions in the TGF-β signalling cascade are associated with several human diseases. SMAD2 is one of ...the principal effectors of TGF-β signalling. It interacts with various transcription factors, and is extensively regulated by post-translational modifications, such as ubiquitination. However, much remains unknown about the regulation of SMAD2 function. Using a proteomic screen, I identified ubiquitin-specific peptidase 11 (USP11) as a novel SMAD2 interactor. I confirmed their mutual interaction, and showed that USP11 specifically interacts with the linker domain of SMAD2, but did not appear to regulate either its stability or ubiquitination pattern. USP11 knockdown decreased TGF-β-mediated gene promoter-reporter activity, whereas USP11 over-expression potentiated it. USP11 knockdown did not, however, affect endogenous gene expression after 2 h TGF-β treatment, as determined by microarray analysis and quantitative reverse transcription PCR. I conclude that although USP11 did not regulate the stability of SMAD2, it might be involved in the regulation of other, currently unresolved, aspects of TGF-β signalling.
La signalisation de TGF-β contrôle des processus cellulaires variés au cours du développement et de l'âge adulte. Des perturbations dans la cascade TGF-β sont associées à plusieurs maladies humaines. SMAD2 est l'un des principaux effecteurs de cette cascade et sa fonction est modulée par des modifications post-traductionnelles comme l'ubiquitination. Cependant, il reste encore plusieurs interrogations à propos de sa modulation. En utilisant une approche protéomique, j'ai identifié la peptidase spécifique de l'ubiquitin -11 (USP11) comme un nouveau partenaire de SMAD2. J'ai confirmé leur interaction et démontré qu'USP11 interagit avec le domaine de liaison de SMAD2, mais n'affecte pas sa stabilité ou son ubiquitination. La suppression d'USP11 par siRNA a diminué l'activité de gène rapporteur de TGF-β, mais n'a pas affecté l'expression des gènes endogènes, comme déterminé à l'aide de puce à ADN et par qPCR. En conclusion, même si USP11 ne contrôle pas la stabilité de SMAD2, il peut être impliqué dans la régulation de la signalisation TGF-β à un autre niveau.
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