Contribution of mutant HSC clones to immature and mature cells in MDS and CMML, and variations with AZA therapy.

Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.

RNA Splicing Alterations Induce a Cellular Stress Response Associated with Poor Prognosis in Acute Myeloid Leukemia.

PURPOSE: RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are common in some hematologic cancers but are relatively uncommon in acute myeloid leukemia (AML, < 20% of patients). We examined whether RNA splicing differences exist in AML, even in the absence of splicing factor mutations. EXPERIMENTAL DESIGN: We developed a bioinformatics pipeline to study alternative RNA splicing in RNA-sequencing data from large cohorts of patients with AML. RESULTS: We have identified recurrent differential alternative splicing between patients with poor and good prognosis. These splicing events occurred even in patients without any discernible splicing factor mutations. Alternative splicing recurrently occurred in genes with specific molecular functions, primarily related to protein translation. Developing tools to predict the functional impact of alternative splicing on the translated protein, we discovered that approximately 45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced and the splicing of their target transcripts were altered. Studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and upregulation of inflammation-related genes. Finally, using machine learning techniques, we identified a splicing signature of four genes which refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. CONCLUSIONS: Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance.See related commentary by Bowman, p. 3503.

Lymphatic Migration of Immune Cells.

Lymphatic vessels collect interstitial fluid that has extravasated from blood vessels and return it to the circulatory system. Another important function of the lymphatic network is to facilitate immune cell migration and antigen transport from the periphery to draining lymph nodes. This migration plays a crucial role in immune surveillance, initiation of immune responses and tolerance. Here we discuss the significance and mechanisms of lymphatic migration of innate and adaptive immune cells in homeostasis, inflammation and cancer.

In vivo photolabeling of tumor-infiltrating cells reveals highly regulated egress of T-cell subsets from tumors.

Immune therapy is rapidly gaining prominence in the clinic as a major weapon against cancer. Whereas much attention has been focused on the infiltration of tumors by immune cells, the subsequent fate of these infiltrates remains largely unexplored. We therefore established a photoconversion-based model that allowed us to label tumor-infiltrating immune cells and follow their migration. Using this system, we identified a population of tumor-experienced cells that emigrate from primary tumors to draining lymph nodes via afferent lymphatic vessels. Although the majority of tumor-infiltrating cells were myeloid, T cells made up the largest population of tumor-egressing leukocytes. Strikingly, the subset composition of tumor-egressing T cells was greatly skewed compared with those that had infiltrated the tumor and those resident in the draining lymph node. Some T-cell subsets such as CD8+ T cells emigrated more readily; others including CD4-CD8- T cells were preferentially retained, suggesting that specific mechanisms guide immune cell egress from tumors. Furthermore, tumor-egressing T cells were more activated and displayed enhanced effector function in comparison with their lymph node counterparts. Finally, we demonstrated that tumor-infiltrating T cells migrate to distant secondary tumors and draining lymph nodes, highlighting a mechanism whereby tumor-experienced effector T cells may mediate antitumor immunity at metastatic sites. Thus, our results provide insights into migration and function of tumor-infiltrating immune cells and the role of these cells in tumor immunity outside of primary tumor deposits.

The lymph node neutrophil.

Secondary lymphoid organs provide a specialized microenvironment tailored to foster communication between cells of the innate and adaptive immune systems. These interactions allow immune cells to coordinate multilayered defense against pathogens. Until recently dendritic cells and macrophages were thought to comprise the main innate immune cell subsets responsible for delivering signals that drive the adaptive immune response, while the function of neutrophils was largely confined to the innate immune system. However, the discovery of neutrophils in lymph nodes has raised the question of whether neutrophils might play a more extensive role not only in innate immunity per se, but also in coordinating the interactions between innate and adaptive immune responses. In this review we discuss the mechanisms and consequences of neutrophil recruitment to lymph nodes and how this recruitment influences subsequent immune responses both in situ and at distant sites.

Microbe-dependent lymphatic migration of neutrophils modulates lymphocyte proliferation in lymph nodes.

Neutrophil recruitment to the site of injury is an essential first step of an anti-bacterial response. However, little is known about the basis for and relevance of neutrophil migration from inflamed tissue into lymphoid organs. We established a photoconversion-based system to monitor the fate of neutrophils recruited to inflamed skin. While neutrophils are efficiently recruited to sites of both microbial and sterile lesions, subsequent re-localization to draining lymph nodes happens only when bacteria are present in the primary lesion. Skin egress of neutrophils occurs via lymphatic vessels and is dependent on CD11b and CXCR4 but not CCR7. Neutrophils are the predominant immune cell to migrate from inflamed skin into lymph nodes where they augment lymphocyte proliferation. Furthermore, inhibition of neutrophil migration from skin reduces T-cell proliferation in draining lymph nodes. Thus neutrophils mediate rapid cellular communication between the initial injury site and secondary lymphoid organs and modulate immune responsiveness.

T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice.

The potential usefulness of intravital two-photon microscopy for fate mapping is limited by its inability to track cells beyond the confines of the imaging volume. Therefore, we have developed and validated a novel method for in vivo photolabelling of spatially-restricted cells expressing the Kaede optical highlighter by two-photon excitation. This has allowed us to optically mark a cohort of follicular B cells and track their dissemination from the original imaging volume in the lymph node to the spleen and contralateral lymph node. We also present the first demonstration, to our knowledge, of in vivo photoconversion of a freely moving single cell in a live adult animal. This method of `discontinuous' cell tracking therefore significantly extends the fate mapping capabilities of two-photon microscopy to delineate the spatiotemporal dynamics of cellular processes that span multiple anatomical sites at the single cell level.

Optimizing fluorescence excitation and detection for intravital two-photon microscopy.

Commercial two-photon microscope systems incorporating turnkey ultrafast lasers have made the technology more user-friendly and accessible to nonspecialized biology laboratories. This has been accompanied by the development of an exciting range of new fluorescent proteins and dyes such as near-infra-red fluorescent proteins and optical highlighters. However, the two-photon absorption properties of these fluorescent molecules are not widely available and cannot be reliably predicted from their single photon absorption spectra. Furthermore, the spectral characteristics of fluorescent proteins in vivo can be affected by the local environment and light scattering by deep tissue and can vary greatly from one laboratory to the next. Here, we describe a simple protocol for determining the two-photon excitation peaks of fluorescent reporters that can be tailored to the relevant tissue samples to suit the imaging goals of individual biological laboratories.