Tissue-Specific Features of the T Cell Repertoire After Allogeneic Hematopoietic Cell Transplantation in Human and Mouse

T cells are the central drivers of many inflammatory diseases, but the repertoire of tissue-resident T cells at sites of pathology in human organs remains poorly understood. We examined the site-specificity of T cell receptor (TCR) repertoires across tissues (5 to 18 tissues per patient) in prospectively collected autopsies of patients with and without graft-versus-host disease (GVHD), a potentially lethal tissue-targeting complication of allogeneic hematopoietic cell transplantation, and in mouse models of GVHD. Anatomic similarity between tissues was a key determinant of TCR repertoire composition within patients, independent of disease or transplant status. The T cells recovered from peripheral blood and spleens in patients and mice captured a limited portion of the TCR repertoire detected in tissues. Whereas few T cell clones were shared across patients, motif-based clustering revealed shared repertoire signatures across patients in a tissue-specific fashion. T cells at disease sites had a tissue-resident phenotype and were of donor origin based on single-cell chimerism analysis. These data demonstrate the complex composition of T cell populations that persist in human tissues at the end stage of an inflammatory disorder after lymphocyte-directed therapy. These findings also underscore the importance of studying T cell in tissues rather than blood for tissue-based pathologies and suggest the tissue-specific nature of both the endogenous and posttransplant T cell landscape

Signalisation par le récepteur de la thrombopoïétine et syndromes myéloprolifératifs non-LMC

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Induction of myeloproliferative disorder and myelofibrosis by thrombopoietin receptor W515 mutants is mediated by cytosolic tyrosine 112 of the receptor

Constitutively active JAK2V617F and thrombopoietin receptor (TpoR) W515L/K mutants are major determinants of human myeloproliferative neoplasms (MPNs). We show that a TpoRW515 mutation (W515A), whichwedetected in 2 myelofibrosis patients, and the Delta 5TpoR active mutant, where the juxtamembrane R/KW515QFP motif is deleted, induce a myeloproliferative phenotype in mouse bone marrow reconstitution experiments. This phenotype required cytosolic Y112 of the TpoR. Phosphotyrosine immunoprofiling detected phosphorylated cytosolic TpoR Y78 and Y112 in cells expressing TpoRW515A. Mutation of cytosolic Y112 to phenylalanine prevented establishment of the in vivo phenotype and decreased constitutive active signaling by Delta 5TpoR and TpoRW515A, especially via the mitogen-activated protein (MAP)-kinase pathway, without decreasing Janus kinase 2 (JAK2) activation. In contrast, mutation of cytosolic Y78 to phenylalanine enhanced the myeloproliferative syndrome induced by the TpoRW515 mutants, by enhancing receptor-induced JAK2 activation. We propose that TpoR cytosolic phosphorylated Y112 and flanking sequences could become targets for pharmacologic inhibition in MPNs. (Blood. 2010;115:1037-1048

High Throughput Droplet Single-Cell Genotyping of Transcriptomes (GoT) Reveals the Cell Identity Dependency of the Transcriptional Output of Somatic Mutations

Abstract Somatic mutations in hematopoietic precursors underlie the development of myeloid disorders, such as myeloproliferative neoplasms (MPN). However, our ability to interrogate the transcriptional impact of these mutations on human hematopoiesis is limited by the frequent admixing of mutant (MUT) with wildtype (WT) cells or with other subclones. Recently, digital single-cell RNA-sequencing has provided high-resolution maps of normal hematopoiesis. Nonetheless, due to their 3' bias, these methods do not capture the cell's mutational status. Efficient linking of single-cell genotype and transcriptomes would allow direct comparison of WT and MUT progenitors within the same sample, eliminating patient-specific and technical confounders. Thus, we developed single-cell Genotyping of Transcriptomes (GoT) to link genotypes of expressed genes to transcriptional profiling of thousands of cells by adapting the 10x Genomics platform. We capture the target locus from cDNA generated at an intermediate step, thus enabling linkage of genotype to whole transcriptomes via shared barcodes (Fig. 1A). We tested this approach via a species-mixing experiment, whereby mouse cells with MUT CALR were mixed with human cells with WT CALR. GoT of 1291 admixed cells provided genotyping for 97.5% of cells, and 96.7% matched the expected species (Fig. 1B). To demonstrate the ability of this technology to probe hematopoietic differentiation in MPN, we applied GoT to 20,908 CD34+ cells across five patients with CALR-mutated essential thrombocythemia (ET) or myelofibrosis (MF), resulting in genotyping of 82% of cells. We first performed clustering agnostic to genotype, based on transcriptome data alone, and found that cells clustered according to progenitor cell identity, rather than mutational status (Fig. 1C). Furthermore, projection of GoT data demonstrated that MUT cells were present across all progenitor clusters (Fig. 1D). However, the frequency of CALR-mutated cells was higher in committed progenitors, especially megakaryocytic progenitors (MkPs) compared to CD34+, CD38- hematopoietic stem progenitor cells (HSCPs, Fig. 1E). Thus, CALR mutation may confer a greater fitness impact in lineage-committed cells vs. HSPCs. Indeed, we found a significant increase in the number of MkPs in cell cycle in MUT cells compared to WT cells (Fig. 1F). Moreover, this increase in cell cycle activity correlated with the platelet count (Fig. 1G). This suggests that interrogation of MUT and WT progenitors may inform our understanding of patient phenotypic variability despite shared genotypes. GoT enables de novo differential expression discovery in MUT vs. WT cells within the same progenitor subset. MUT MkPs upregulated genes in the unfolded protein response, such as PDIA6, HSPA5 and XBP1 (Fig. 1H), consistent with the central role of CALR as a chaperone protein. On the other hand, MUT HSPCs showed upregulation of the NF-kB pathway (Fig. 1I), most significantly in the subcluster enriched with the earliest HSCs (Fig. 1J). Since the NF-kB pathway has been implicated in HSC self-renewal, our data provides a potential mechanism for clonal expansion and maintenance of CALR-mutated HSCs. Collectively, these findings demonstrate that the transcriptomic output of CALR mutations is closely dependent on cell identity. To further evaluate the potential of GoT to detect multiple genotypes in clonally complex neoplasms, we targeted three genes, clonal SF3B1 (VAF 47.5% by bulk exon sequencing), and subclonal CALR (43.5%) and NFE2 (33%), in CD34+ cells from a patient with MF (Fig. 1K). Through GoT, the subclonal transcriptional output was interrogated; for example, CALR mutation conferred proliferative advantage to megakaryocytic-erythroid progenitors even in the presence of SF3B1 mutation, while additional NFE2 mutation did not further increase cell cycle activity. In summary, GoT is a powerful tool for linking transcriptional changes to somatic genotypes at the single-cell level. Specifically, it uncovered the transcriptional impact of mutations in myeloid clonal growths in the context of distinct progenitor identities. Further application of GoT to additional MPN contexts as well as clonal hematopoiesis is thus anticipated to provide critical insights into the transcriptional programs that enable clonal expansion and evolution in human hematopoiesis. Disclosures Hoffman: Merus: Research Funding; Janssen: Research Funding; Summer Road: Research Funding; Incyte: Research Funding; Formation Biologics: Research Funding

Discovery of Candidate DNA Methylation Cancer Driver Genes

Epigenetic alterations, such as promoter hypermethylation, may drive cancer through tumor suppressor gene inactivation. However, we have limited ability to differentiate driver DNA methylation (DNAme) changes from passenger events. We developed DNAme driver inference-MethSig-accounting for the varying stochastic hypermethylation rate across the genome and between samples. We applied MethSig to bisulfite sequencing data of chronic lymphocytic leukemia (CLL), multiple myeloma, ductal carcinoma in situ, glioblastoma, and to methylation array data across 18 tumor types in TCGA. MethSig resulted in well-calibrated quantile-quantile plots and reproducible inference of likely DNAme drivers with increased sensitivity/specificity compared with benchmarked methods. CRISPR/Cas9 knockout of selected candidate CLL DNAme drivers provided a fitness advantage with and without therapeutic intervention. Notably, DNAme driver risk score was closely associated with adverse outcome in independent CLL cohorts. Collectively, MethSig represents a novel inference framework for DNAme driver discovery to chart the role of aberrant DNAme in cancer. SIGNIFICANCE: MethSig provides a novel statistical framework for the analysis of DNA methylation changes in cancer, to specifically identify candidate DNA methylation driver genes of cancer progression and relapse, empowering the discovery of epigenetic mechanisms that enhance cancer cell fitness

Somatic mutations and cell identity linked by Genotyping of Transcriptomes

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34 cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity