3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Nanoparticle-based modulation of CD4+ T cell effector and helper functions enhances adoptive immunotherapy.

Helper (CD4+) T cells perform direct therapeutic functions and augment responses of cells such as cytotoxic (CD8+) T cells against a wide variety of diseases and pathogens. Nevertheless, inefficient synthetic technologies for expansion of antigen-specific CD4+ T cells hinders consistency and scalability of CD4+ T cell-based therapies, and complicates mechanistic studies. Here we describe a nanoparticle platform for ex vivo CD4+ T cell culture that mimics antigen presenting cells (APC) through display of major histocompatibility class II (MHC II) molecules. When combined with soluble co-stimulation signals, MHC II artificial APCs (aAPCs) expand cognate murine CD4+ T cells, including rare endogenous subsets, to induce potent effector functions in vitro and in vivo. Moreover, MHC II aAPCs provide help signals that enhance antitumor function of aAPC-activated CD8+ T cells in a mouse tumor model. Lastly, human leukocyte antigen class II-based aAPCs expand rare subsets of functional, antigen-specific human CD4+ T cells. Overall, MHC II aAPCs provide a promising approach for harnessing targeted CD4+ T cell responses.

Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods.

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4+ T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

Complex decay dynamics of HIV virions, intact and defective proviruses, and 2LTR circles following initiation of antiretroviral therapy.

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4+ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4+ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5' or 3' end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

Mouse Homologue of Human HLA-DO Does Not Preempt Autoimmunity but Controls Murine Gammaherpesvirus MHV68.

H2-O (human HLA-DO) is a relatively conserved nonclassical MHC class II (MHCII)-like molecule. H2-O interaction with human HLA-DM edits the repertoire of peptides presented to TCRs by MHCII. It was long hypothesized that human HLA-DM inhibition by H2-O provides protection from autoimmunity by preventing binding of the high-affinity self-peptides to MHCII. The available evidence supporting this hypothesis, however, was inconclusive. A possibility still remained that the effect of H2-O deficiency on autoimmunity could be better revealed by using H2-O-deficient mice that were already genetically predisposed to autoimmunity. In this study, we generated and used autoimmunity-prone mouse models for systemic lupus erythematosus and organ-specific autoimmunity (type 1 diabetes and multiple sclerosis) to definitively test whether H2-O prevents autoimmune pathology. Whereas our data failed to support any significance of H2-O in protection from autoimmunity, we found that it was critical for controlling a γ-herpesvirus, MHV68. Thus, we propose that H2-O editing of the MHCII peptide repertoire may have evolved as a safeguard against specific highly prevalent viral pathogens.

Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus.

Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein1 to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex2-4. Although recent studies have observed the presence of the capsid protein in the nucleus during infection5-8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.