Protocol for analysis of single-cell sequencing data by Seqtometry.

Seqtometry (sequencing-to-measurement) is an analytical platform for single-cell analysis based on direct profiling of gene expression and accessibility achieved by advanced scoring with gene signatures. Here, we present a protocol for single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) analysis using Seqtometry. We describe steps for preprocessing, imputation, scoring, and plotting, with extensions to large datasets and integration of multiple datasets.

Episomal Vectors for Stable Production of Recombinant Proteins and Engineered Antibodies.

There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest.

Emerging T cell immunoregulatory mechanisms in multiple sclerosis and Alzheimer's disease.

Multiple sclerosis (MS) and Alzheimer's disease (AD) are neuroinflammatory and neurodegenerative diseases with considerable socioeconomic impacts but without definitive treatments. AD and MS have multifactorial pathogenesis resulting in complex cognitive and neurologic symptoms and growing evidence also indicates key functions of specific immune cells. Whereas relevant processes dependent on T cells have been elucidated in both AD and MS, mechanisms that can control such immune responses still remain elusive.

EPISOMAL VECTORS FOR STABLE PRODUCTION OF RECOMBINANT PROTEINS AND ENGINEERED ANTIBODIES.

There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance though an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest.

Single-cell sequencing analysis within biologically relevant dimensions.

The currently predominant approach to transcriptomic and epigenomic single-cell analysis depends on a rigid perspective constrained by reduced dimensions and algorithmically derived and annotated clusters. Here, we developed Seqtometry (sequencing-to-measurement), a single-cell analytical strategy based on biologically relevant dimensions enabled by advanced scoring with multiple gene sets (signatures) for examination of gene expression and accessibility across various organ systems. By utilizing information only in the form of specific signatures, Seqtometry bypasses unsupervised clustering and individual annotations of clusters.

Stimulation of Immune Checkpoint Molecule B and T-Lymphocyte Attenuator Alleviates Experimental Crescentic Glomerulonephritis.

Significance Statement: Treatment of acute, crescentic glomerulonephritis (GN) consists of unspecific and potentially toxic immunosuppression. T cells are central in the pathogenesis of GN, and various checkpoint molecules control their activation. The immune checkpoint molecule B and T-lymphocyte attenuator (BTLA) has shown potential for restraining inflammation in other T-cell-mediated disease models.

Activation, Amplification, and Ablation as Dynamic Mechanisms of Dendritic Cell Maturation.

T cell responses to cognate antigens crucially depend on the specific functionality of dendritic cells (DCs) activated in a process referred to as maturation. Maturation was initially described as alterations of the functional status of DCs in direct response to multiple extrinsic innate signals derived from foreign organisms. More recent studies, conducted mainly in mice, revealed an intricate network of intrinsic signals dependent on cytokines and various immunomodulatory pathways facilitating communication between individual DCs and other cells for the orchestration of specific maturation outcomes.

Life and death of tolerogenic dendritic cells.

In contrast to conventional dendritic cells (cDCs) that are constantly exposed to microbial signals at anatomical barriers, cDCs in systemic lymphoid organs are sheltered from proinflammatory stimulation in the steady state but respond to inflammatory signals by gaining specific immune functions in a process referred to as maturation. Recent findings show that, during maturation, a population of systemic tolerogenic cDCs undergoes an acute tumor necrosis factor α (TNFα)-mediated cell death, resulting in the loss of tolerance-inducing capacity. This tolerogenic cDC population is restored upon return to the homeostatic baseline.

Roles of Hopx in the differentiation and functions of immune cells.

Homeodomain only protein (Hopx, HOPX) is a highly evolutionarily conserved, homeodomain-containing, small protein expressed in multiple tissues and cell types, including those of hematopoietic origin. The quasi-ubiquitous presence of Hopx contrasts with its specialized and context-dependent roles in various cell lineages. Recently, versatile functions of Hopx have been revealed in immune cells, including T lymphocytes with effector and regulatory roles.

TNF-α sculpts a maturation process in vivo by pruning tolerogenic dendritic cells.

It remains unclear how the pro-immunogenic maturation of conventional dendritic cells (cDCs) abrogates their tolerogenic functions. Here, we report that the loss of tolerogenic functions depends on the rapid death of BTLA cDC1s, which, in the steady state, are present in systemic peripheral lymphoid organs and promote tolerance that limits subsequent immune responses. A canonical inducer of maturation, lipopolysaccharide (LPS), initiates a burst of tumor necrosis factor alpha (TNF-α) production and the resultant acute death of BTLA cDC1s mediated by tumor necrosis factor receptor 1.

Applications of Antibody-Based Antigen Delivery Targeted to Dendritic Cells In Vivo.

Recombinant immunoglobulins, derived from monoclonal antibodies recognizing the defined surface epitopes expressed on dendritic cells, have been employed for the past two decades to deliver antigens to dendritic cells in vivo, serving as critical tools for the investigation of the corresponding T cell responses. These approaches originated with the development of the recombinant chimeric antibody against a multilectin receptor, DEC-205, which is present on subsets of murine and human conventional dendritic cells. Following the widespread application of antigen targeting through DEC-205, similar approaches then utilized other epitopes as entry points for antigens delivered by specific antibodies to multiple types of dendritic cells.

Variegated Outcomes of T Cell Activation by Dendritic Cells in the Steady State.

Conventional dendritic cells (cDC) control adaptive immunity by sensing damage- and pathogen-associated molecular patterns and then inducing defined differentiation programs in T cells. Nevertheless, in the absence of specific proimmunogenic innate signals, generally referred to as the steady state, cDC also activate T cells to induce specific functional fates. Consistent with the maintenance of homeostasis, such specific outcomes of T cell activation in the steady state include T cell clonal anergy, deletion, and conversion of peripheral regulatory T cells (pTregs).

Landscape of Hopx expression in cells of the immune system.

Homeodomain only protein (Hopx) is a regulator of cell differentiation and function, and it has also emerged as a crucial marker of specific developmental and differentiation potentials. Hopx expression and functions have been identified in some stem cells, tumors, and in certain immune cells. However, expression of Hopx in immune cells remains insufficiently characterized.

Current and Future Immunotherapies for Multiple Sclerosis.

Despite substantial progress in developing new immunotherapies against multiple sclerosis (MS), currently available immunotherapies are only partially effective for this debilitating neurological disease, thus necessitating new therapeutic approaches. Here, we review the immunotherapies already approved for MS as well as relevant clinical trials. Further, we present some experimental approaches that are currently being developed and are focused on modulating the functions of dendritic cells and regulatory T cells.

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown.

A Two-Step Process of Effector Programming Governs CD4 T Cell Fate Determination Induced by Antigenic Activation in the Steady State.

Various processes induce and maintain immune tolerance, but effector T cells still arise under minimal perturbations of homeostasis through unclear mechanisms. We report that, contrary to the model postulating primarily tolerogenic mechanisms initiated under homeostatic conditions, effector programming is an integral part of T cell fate determination induced by antigenic activation in the steady state. This effector programming depends on a two-step process starting with induction of effector precursors that express Hopx and are imprinted with multiple instructions for their subsequent terminal effector differentiation.

Targeting Dendritic Cells with Antigen-Delivering Antibodies for Amelioration of Autoimmunity in Animal Models of Multiple Sclerosis and Other Autoimmune Diseases.

The specific targeting of dendritic cells (DCs) using antigen-delivering antibodies has been established to be a highly efficient protocol for the induction of tolerance and protection from autoimmune processes in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), as well as in some other animal disease models. As the specific mechanisms of such induced tolerance are being investigated, the newly gained insights may also possibly help to design effective treatments for patients. Here we review approaches applied for the amelioration of autoimmunity in animal models based on antibody-mediated targeting of self-antigens to DCs.

Natural and Induced Tolerogenic Dendritic Cells.

Dendritic cells (DCs) are highly susceptible to extrinsic signals that modify the functions of these crucial APCs. Maturation of DCs induced by diverse proinflammatory conditions promotes immune responses, but certain signals also induce tolerogenic functions in DCs. These "induced tolerogenic DCs" help to moderate immune responses such as those to commensals present at specific anatomical locations.

Advancing immunomodulation by in vivo antigen delivery to DEC-205 and other cell surface molecules using recombinant chimeric antibodies.

A targeted delivery of defined antigens in vivo allows for the probing of relevant functions of the immune system. Recombinant chimeric antibodies, produced by genetically modifying original monoclonal antibodies specific for molecules expressed on dendritic cells and other immune cells, have paved the way for the development of such strategies and have become reliable tools for achieving a specific immunomodulation. These antibodies have proven important in both basic research and clinical applications, extending data obtained in disease models of autoimmunity and cancer.

Immunomodulatory Bonds of the Partnership between Dendritic Cells and T Cells.

By acquiring, processing, and presenting both foreign and self-antigens, dendritic cells (DCs) initiate T cell activation that is shaped through the immunomodulatory functions of a variety of cell-membrane-bound molecules including BTLA-HVEM, CD40-CD40L, CTLA-4-CD80/CD86, CD70-CD27, ICOS-ICOS-L, OX40-OX40L, and PD-L1-PD-1, as well as several key cytokines and enzymes such as interleukin-6 (IL-6), IL-12, IL-23, IL-27, transforming growth factor-beta 1 (TGF-β1), retinaldehyde dehydrogenase (Raldh), and indoleamine 2,3-dioxygenase (IDO). Some of these distinct immunomodulatory signals are mediated by specific subsets of DCs, therefore contributing to the functional specialization of DCs in the priming and regulation of immune responses. In addition to responding to the DC-mediated signals, T cells can reciprocally modulate the immunomodulatory capacities of DCs, further refining immune responses.

Dendritic Cells As Inducers of Peripheral Tolerance.

Mechanisms of tolerance initiated in the thymus are indispensable for establishing immune homeostasis, but they may not be sufficient to prevent tissue-specific autoimmune diseases. In the periphery, dendritic cells (DCs) play a crucial tolerogenic role, extending the maintenance of immune homeostasis and blocking autoimmune responses. We review here these essential roles of DCs in orchestrating mechanisms of peripheral T cell tolerance as determined by targeted delivery of defined antigens to DCs in vivo in combination with various genetic modifications of DCs.

TLR7 Signaling Regulates Th17 Cells and Autoimmunity: Novel Potential for Autoimmune Therapy.

Innate regulation through TLR signaling has been shown to be important for promoting T cell subset development and function. However, limited information is known about whether differential TLR signaling can selectively inhibit Th17 and/or Th1 cells, which are important for controlling excessive inflammation and autoimmune responses. In this article, we demonstrate that activation of TLR7 signaling in T cells can inhibit Th17 cell differentiation from naive T cells and IL-17 production in established Th17 cells.

Peripherally Induced Regulatory T Cells: Recruited Protectors of the Central Nervous System against Autoimmune Neuroinflammation.

Defects in regulatory T cells (Treg cells) aggravate multiple sclerosis (MS) after its onset and the absence of Treg cell functions can also exacerbate the course of disease in an animal model of MS. However, autoimmune neuroinflammation in many MS models can be acutely provoked in healthy animals leading to an activation of encephalitogenic T cells despite the induction of immune tolerance in the thymus including thymically produced (t)Treg cells. In contrast, neuroinflammation can be ameliorated or even completely prevented by the antigen-specific Treg cells formed extrathymically in the peripheral immune system (pTreg cells) during tolerogenic responses to relevant neuronal antigens.