Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft versus Leukemia Response with Adoptive Cellular Therapy.

Understanding how intra-tumoral immune populations coordinate to generate anti-tumor responses following therapy can guide precise treatment prioritization. We performed systematic dissection of an established adoptive cellular therapy, donor lymphocyte infusion (DLI), by analyzing 348,905 single-cell transcriptomes from 74 longitudinal bone-marrow samples of 25 patients with relapsed myeloid leukemia; a subset was evaluated by protein-based spatial analysis. In acute myelogenous leukemia (AML) responders, diverse immune cell types within the bone-marrow microenvironment (BME) were predicted to interact with a clonally expanded population of ZNF683 + GZMB + CD8+ cytotoxic T lymphocytes (CTLs) which demonstrated in vitro specificity for autologous leukemia. This population, originating predominantly from the DLI product, expanded concurrently with NK and B cells. AML nonresponder BME revealed a paucity of crosstalk and elevated TIGIT expression in CD8+ CTLs. Our study highlights recipient BME differences as a key determinant of effective anti-leukemia response and opens new opportunities to modulate cell-based leukemia-directed therapy.

Integrative genotyping of cancer and immune phenotypes by long-read sequencing.

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 longitudinal tracking of subclones of acute myeloid leukemia (AML) and describe the heterogeneous isoform landscape of thousands of marrow-infiltrating immune cells. We propose that enhanced cellular genotyping using nanoranger can improve the tracking of single-cell tumor and immune cell co-evolution.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

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 syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection 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, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

Spatial maps of T cell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response.

T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their clonotypic T cell receptors (TCRs). Determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell behavior, but spatial sequencing methods remain unable to profile the TCR repertoire. Here, we developed Slide-TCR-seq, a 10-µm-resolution method, to sequence whole transcriptomes and TCRs within intact tissues. We confirmed the ability of Slide-TCR-seq to map the characteristic locations of T cells and their receptors in mouse spleen. In human lymphoid germinal centers, we identified spatially distinct TCR repertoires. Profiling T cells in renal cell carcinoma and melanoma specimens revealed heterogeneous immune responses: T cell states and infiltration differed intra- and inter-clonally, and adjacent tumor and immune cells exhibited distinct gene expression. Altogether, our method yields insights into the spatial relationships between clonality, neighboring cell types, and gene expression that drive T cell responses.

High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria.

As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO2-concentrating mechanism (CCM) to impose a high-CO2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. We showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 108 CFU.