Demo Faculty

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting TIM-3 for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab-treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment-resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ T cells (Tc) enhanced Tc activation, proliferation and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3-treatment …

Achieving long-term disease control using therapeutic immunomodulation is a long-standing concept with a strong tradition in blood malignancies. Besides allogeneic hematopoietic stem cell transplantation that continues to provide potentially curative treatment for otherwise challenging diagnoses, recent years have seen impressive progress in immunotherapies for leukemias and lymphomas with immune checkpoint blockade, bispecific monoclonal antibodies, and CAR T cell therapies. Despite their success, non-response, relapse, and immune toxicities remain frequent, thus prioritizing the elucidation of the underlying mechanisms and identifying predictive biomarkers. The increasing availability of single-cell genomic tools now provides a system’s immunology view to resolve the molecular and cellular mechanisms of immunotherapies at unprecedented resolution. Here, we review recent studies that leverage …

BackgroundPredictive biomarkers of immune checkpoint inhibitor (ICI) efficacy are currently lacking for non-small cell lung cancer (NSCLC). Here, we describe the results from the Anti–PD-1 Response Prediction DREAM Challenge, a crowdsourced initiative that enabled the assessment of predictive models by using data from two randomized controlled clinical trials (RCTs) of ICIs in first-line metastatic NSCLC.MethodsParticipants developed and trained models using public resources. These were evaluated with data from the CheckMate 026 trial (NCT02041533), according to the model-to-data paradigm to maintain patient confidentiality. The generalizability of the models with the best predictive performance was assessed using data from the CheckMate 227 trial (NCT02477826). Both trials were phase III RCTs with a chemotherapy control arm, which supported the differentiation between predictive and prognostic …

Single-cell transcriptomics has become the definitive method for classifying cell types and states, and can be augmented with genotype information to improve cell lineage identification. Due to constraints of short-read sequencing, current methods to detect natural genetic barcodes often require cumbersome primer panels and early commitment to targets. Here we devise a flexible long-read sequencing workflow and analysis pipeline, termed nanoranger, that starts from intermediate single-cell cDNA libraries to detect cell lineage-defining features, including single-nucleotide variants, fusion genes, isoforms, sequences of chimeric antigen and TCRs. Through systematic analysis of these classes of natural ‘barcodes’, we define the optimal targets for nanoranger, namely those loci close to the 5’ end of highly expressed genes with transcript lengths shorter than 4 kB. As proof-of-concept, we apply nanoranger to …

Recent spatial transcriptomics (ST) technologies have enabled sub-single-cell resolution profiling of gene expression across the whole transcriptome. However, the transition to high-definition ST significantly increased sparsity and dimensionality, posing computational challenges in discerning cell identities, understanding neighborhood structure, and identifying differential expression - all are crucial steps to study normal and disease ST samples. Here we present STHD, a novel machine learning method for probabilistic cell typing of single spots in whole-transcriptome, high-resolution ST data. Unlike current binning-aggregation-deconvolution strategy, STHD directly models gene expression at single-spot level to infer cell type identities. It addresses sparsity by modeling count statistics, incorporating neighbor similarities, and leveraging reference single-cell RNA-seq data. We demonstrated that STHD accurately predicts cell type identities at single-spot level, which automatically achieved precise segmentation of global tissue architecture and local multicellular neighborhoods. The STHD labels facilitated various downstream analyses, including cell type-stratified bin aggregation, spatial compositional comparison, and cell type-specific differential expression analyses. These high-resolution labels further defined frontlines of inter-cell type interactions, revealing direct cell-cell communication activities at immune hubs of a colon cancer sample. Overall, computational modeling of high-resolution spots with STHD uncovers precise spatial organization and deeper biological insights for disease mechanisms.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice, and recapitulated the process of transformation from indolent CLL into large cell lymphoma (i.e. Richter’s syndrome [RS]). Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed co-selection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, down-modulation of the receptor …

Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize …

The therapeutic effect of allo-HSCT relies on T cell alloreactivity driven by genetic nonidentity between donor and recipient (D-R) pairs. In the setting of HLA-matched transplants, endogenous proteins in recipient cells differing from those of the donor due to genetic polymorphisms, can provide distinct HLA-binding peptides, serving as minor histocompatibility antigens (mHAgs). Hematopoietically-restricted mHAgs represent ideal targets to separate graft-versus-leukemia (GvL) from graft-versus-host-disease (GvHD) effects. To date,however, the quest for optimal GvL mHAgs has resulted in the identification of only a handful of targets, raising concerns on the clinical feasibility of their targeting.We hypothesized that systematic mHAg prediction from whole-exome sequencing (WES) of D-R pairs could help identifying novel candidate mHAgs with an acceptable safety profile for therapeutical targeting. We focused on …

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow.To infer cell-cell interactions in a …

T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms (SNPs, indels, frameshifts), is at the core of the therapeutic effect of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite the crucial role of mHAgs in graft-versus-leukemia and graft-versus-host (GvHD) reactions, it has not been possible thus far to consistently link patient-specific mHAg repertoires to clinical outcomes. As a result, only D-R HLA matching and the activity of GvHD prophylaxis strategies are currently available to help clinicians in this challenge.We hypothesized that post-transplant outcomes could be impacted by genome-wide mHAg load, delineated in an organ- and malignancy-specific fashion. We have therefore devised an analytic framework to systematically identify autosomal and Y-encoded mHAgs, based …

IntroductionEfforts to enhance T cell-mediated acute myeloid leukemia (AML) eradication face challenges possibly due to unique mechanisms of leukemia-induced T-cell dysfunction compared to exhaustion observed in solid tumors(Penter et al., Blood 2023). We previously showed the efficacy of Wilms' Tumor (WT) Antigen 1-specific T-cell receptor (TCR C4) inserted into CD8 + T cells (T TCR-C4) in preventing AML recurrence after hematopoietic cell transplantation (HCT) (Chapuis et al., Nat Med 2019). This led us to investigate the safety and efficacy of T TCR-C4 in post-HCT relapsed patients.MethodsIn this Phase I/II study conducted at Fred Hutchinson Cancer Center, 15 patients were enrolled and prospectively monitored for disease response. We manufactured T TCR-C4 using a lentiviral transduction system (Chapuis et al, Nat Med 2019) and performed comprehensive analyses of bone marrow (BM) and …

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow.To infer cell-cell interactions in a …

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay.To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA …

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay.To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA …

Activity of ipilimumab in bone marrow-involved AML has been modest compared to extramedullary AML relapse or solid tumors like melanoma. Single cell transcriptomics (scRNA-seq) could provide deeper understanding of these differences, yet leukemia and immune cell phenotypes defined by somatic mutations or differential isoform expression often cannot be resolved with native short-read scRNA-seq data due to insufficient read coverage. To overcome this limitation, we devised a workflow ( nanoranger) that leverages improved accuracy of long-read sequencing (Oxford Nanopore, ONT) and enables targeted read-out of molecular features including single-nucleotide variants, gene fusions, isoforms, or T cell receptor (TCR) sequences from intermediate scRNA-seq cDNA libraries.To determine the performance of nanoranger, we undertook a series of assessments. First, we established its genotyping accuracy …

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a'cold'tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with response to ipilimumab-based therapy. Altogether, our study provides immune correlates of response to T cell-directed immunotherapies and indicates starting points for further investigations into immunomodulatory mechanisms in AML.

Because of the low mutational burden, children with acute myeloid leukemia (AML) are thought to have a ‘cold’tumor microenvironment and consequently, a low likelihood of response to T cell-directed immunotherapies. Here, we provide a multidimensional overview of the tumor immune microenvironment in newly diagnosed pediatric AML. On a cohort level, we demonstrate wide variation in T cell infiltration with nearly one-third of cases harboring an immune-infiltrated bone marrow. These immune-infiltrated cases are characterized by a decreased abundance of M2-like macrophages, which we find to be associated with response to T cell-directed immunotherapy in adult AML. On an organizational level, we reveal the composition of spatially organized immune aggregates in pediatric AML, and show that in the adult setting such aggregates in post-treatment bone marrow and extramedullary sites associate with …

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti–programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating …

Our genomes influence nearly every aspect of human biology from molecular and cellular functions to phenotypes in health and disease. Human genetics studies have now associated hundreds of thousands of differences in our DNA sequence ("genomic variation") with disease risk and other phenotypes, many of which could reveal novel mechanisms of human biology and uncover the basis of genetic predispositions to diseases, thereby guiding the development of new diagnostics and therapeutics. Yet, understanding how genomic variation alters genome function to influence phenotype has proven challenging. To unlock these insights, we need a systematic and comprehensive catalog of genome function and the molecular and cellular effects of genomic variants. Toward this goal, the Impact of Genomic Variation on Function (IGVF) Consortium will combine approaches in single-cell mapping, genomic perturbations, and predictive modeling to investigate the relationships among genomic variation, genome function, and phenotypes. Through systematic comparisons and benchmarking of experimental and computational methods, we aim to create maps across hundreds of cell types and states describing how coding variants alter protein activity, how noncoding variants change the regulation of gene expression, and how both coding and noncoding variants may connect through gene regulatory and protein interaction networks. These experimental data, computational predictions, and accompanying standards and pipelines will be integrated into an open resource that will catalyze community efforts to explore genome function and the impact …