Response to self antigen imprints regulatory memory in tissues.

Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (T(reg) cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated T(reg) cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, T(reg) cells function to confer 'regulatory memory' to the target tissue. These findings provide a framework for understanding how T(reg) cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.

Cutting edge: Self-antigen controls the balance between effector and regulatory T cells in peripheral tissues.

Immune homeostasis in peripheral tissues is achieved by maintaining a balance between pathogenic effector T cells (Teffs) and protective Foxp3(+) regulatory T cells (Tregs). Using a mouse model of an inducible tissue Ag, we demonstrate that Ag persistence is a major determinant of the relative frequencies of Teffs and Tregs. Encounter of transferred naive CD4(+) T cells with transiently expressed tissue Ag leads to generation of cytokine-producing Teffs and peripheral Tregs. Persistent expression of Ag, a mimic of self-antigen, leads to functional inactivation and loss of the Teffs with preservation of Tregs in the target tissue. The inactivation of Teffs by persistent Ag is associated with reduced ERK phosphorylation, whereas Tregs show less reduction in ERK phosphorylation and are relatively resistant to ERK inhibition. Our studies reveal a crucial role for Ag in maintaining appropriate ratios of Ag-specific Teffs to Tregs in tissues.

Cutting edge: ABIN-1 protects against psoriasis by restricting MyD88 signals in dendritic cells.

Psoriasis is a chronic, inflammatory skin disease caused by a combination of environmental and genetic factors. The Tnip1 gene encodes A20 binding and inhibitor of NF-κB-1 (ABIN-1) protein and is strongly associated with susceptibility to psoriasis in humans. ABIN-1, a widely expressed ubiquitin-binding protein, restricts TNF- and TLR-induced signals. In this study, we report that mice lacking ABIN-1 specifically in dendritic cells (DCs), ABIN-1(fl) CD11c-Cre mice, exhibit perturbed immune homeostasis. ABIN-1-deficient DCs display exaggerated NF-κB and MAPK signaling and produce more IL-23 than do normal cells in response to TLR ligands. Challenge of ABIN-1(fl) CD11c-Cre mice with topical TLR7 ligand leads to greater numbers of Th17 and TCRγδ T cells and exacerbated development of psoriaform lesions. These phenotypes are reversed by DC-specific deletion of the TLR adaptor MyD88. These studies link ABIN-1 with IL-23 and IL-17, and they provide cellular and molecular mechanisms by which ABIN-1 regulates susceptibility to psoriasis.

An immune-based tool platform for in vivo cell clearance.

Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.

Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity.

Partial pluripotent reprogramming can reverse features of aging in mammalian cells, but the impact on somatic identity and the necessity of individual reprogramming factors remain unknown. Here, we used single-cell genomics to map the identity trajectory induced by partial reprogramming in multiple murine cell types and dissected the influence of each factor by screening all Yamanaka Factor subsets with pooled single-cell screens. We found that partial reprogramming restored youthful expression in adipogenic and mesenchymal stem cells but also temporarily suppressed somatic identity programs. Our pooled screens revealed that many subsets of the Yamanaka Factors both restore youthful expression and suppress somatic identity, but these effects were not tightly entangled. We also found that a multipotent reprogramming strategy inspired by amphibian regeneration restored youthful expression in myogenic cells. Our results suggest that various sets of reprogramming factors can restore youthful expression with varying degrees of somatic identity suppression. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Opposing functions of IL-2 and IL-7 in the regulation of immune responses.

Regulation of the magnitude and quality of immune responses is dependent on the integration of multiple signals which typically operate through positive and negative feedback loops. Cytokines that promote or limit T cell expansion and differentiation are often both present in the complex lymphoid environment where antigen-initiated T cell responses take place. The nature and strength of the cytokine signal received by the responding cell, as well as by surrounding regulatory cells, will determine the extent of clonal expansion and the progression towards effector and memory cell differentiation. The mechanisms that determine how much cytokine is produced and how cytokine activities are controlled by receptor expression and intracellular regulators of signaling are not fully understood. Here we discuss the opposing functions of two members of the common receptor gamma chain (γc) cytokines, IL-2 and IL-7 in the generation and regulation of immune responses in vivo.

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.

Inhibition of longevity regulator PAPP-A modulates tissue homeostasis via restraint of mesenchymal stromal cells.

Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloprotease that increases insulin-like growth factor (IGF) availability by cleaving IGF-binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP-A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP-A's role, we developed a PAPP-A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti-PAPP-A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP-A in bone marrow and primary responders to PAPP-A inhibition. Mice treated with anti-PAPP-A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM-producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP-A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC-like cells are the major source of PAPP-A in other tissues also, suggesting that reduced MSC-like cell activity drives the system-wide reduction in ECM gene expression due to PAPP-A inhibition. Dysregulated ECM production is associated with aging and drives age-related diseases, and thus, this may be a mechanism by which PAPP-A deficiency enhances longevity.

Alzheimer's Patient Microglia Exhibit Enhanced Aging and Unique Transcriptional Activation.

Damage-associated microglia (DAM) profiles observed in Alzheimer's disease (AD)-related mouse models reflect an activation state that could modulate AD risk or progression. To learn whether human AD microglia (HAM) display a similar profile, we develop a method for purifying cell types from frozen cerebrocortical tissues for RNA-seq analysis, allowing better transcriptome coverage than typical single-nucleus RNA-seq approaches. The HAM profile we observe bears little resemblance to the DAM profile. Instead, HAM display an enhanced human aging profile, in addition to other disease-related changes such as APOE upregulation. Analyses of whole-tissue RNA-seq and single-cell/nucleus RNA-seq datasets corroborate our findings and suggest that the lack of DAM response in human microglia occurs specifically in AD tissues, not other neurodegenerative settings. These results, which can be browsed at http://research-pub.gene.com/BrainMyeloidLandscape, provide a genome-wide picture of microglial activation in human AD and highlight considerable differences between mouse models and human disease.

Fully Phased Sequence of a Diploid Human Genome Determined de Novo from the DNA of a Single Individual.

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.

Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.

Treating human autoimmunity: current practice and future prospects.

Autoimmune diseases are caused by immune cells attacking the host tissues they are supposed to protect. Recent advances suggest that maintaining a balance of effector and regulatory immune function is critical for avoiding autoimmunity. New therapies, including costimulation blockade, regulatory T cell therapy, antigen-specific immunotherapy, and manipulating the interleukin-2 pathway, attempt to restore this balance. This review discusses these advances as well as the challenges that must be overcome to target these therapies to patients suffering from autoimmune disease while avoiding the pitfalls of general immunosuppression.