Distinctive transcriptomic and epigenomic signatures of bone marrow-derived myeloid cells and microglia in CNS autoimmunity.

In the context of autoimmunity, myeloid cells of the central nervous system (CNS) constitute an ontogenically heterogeneous population that includes yolk sac-derived microglia and infiltrating bone marrow-derived cells (BMC). We previously identified a myeloid cell subset in the brain and spinal cord that expresses the surface markers CD88 and CD317 and is associated with the onset and persistence of clinical disease in the murine model of the human CNS autoimmune disorder, experimental autoimmune encephalomyelitis (EAE). We employed an experimental platform utilizing single-cell transcriptomic and epigenomic profiling of bone marrow-chimeric mice to categorically distinguish BMC from microglia during CNS autoimmunity. Analysis of gene expression and chromosomal accessibility identified CD88+CD317+ myeloid cells in the CNS of EAE mice as originating from BMC and microglia. Interestingly, each cell lineage exhibited overlapping and unique gene expression patterns and transcription factor motifs that allowed their segregation. Our observations will facilitate determining pathogenic contributions of BMC and microglia in CNS autoimmune disease. Ultimately, this agnostic characterization of myeloid cells will be required for devising disease stage-specific and tissue-specific interventions for CNS inflammatory and neurodegenerative disorders.

Serum Neurofilament Light-Chain Concentrations in Essential Tremor: a Case-Control Study.

Essential tremor (ET) is a common neurological disorder, with clinical and pathophysiological links to the cerebellum. Inquiries into the etiology, pathophysiology, and nosology of ET stand to benefit from the identification of disease biomarkers. Serum neurofilament light chain (NfL) has emerged as a novel signature of conditions in which neuronal injury reflects an outcome of the ongoing disease process. We sought to investigate the concentrations of NfL in ET patients and healthy controls. In this case-control study, our powered study population of 41 ET patients and 40 age-matched healthy controls underwent clinical assessments and measurement of serum NfL concentration using Simoa technology. Serum NfL was elevated in ET patients - mean log-transformed serum NfL concentration = 1.23 ± 0.19 (95% confidence interval [CI] = 1.17-1.29) vs. 1.08 ± 0.15 (95% CI = 1.03-1.13), p = 0.0002. This difference persisted after accounting for age, sex and Montreal Cognitive Assessment score in a multiple linear regression model (p = 0.002) and in an age-matched sample subset of 35 ET cases and 35 controls (p = 0.006). There was no association between tremor severity and serum NfL levels (p = 0.73). In this sample of ET patients and controls, serum NfL concentrations were significantly higher in ET. Studies in additional cohorts of ET cases would be of value in attempting to replicate these results and assessing diagnostic utility.

Systems Approaches to Unravel T Cell Function and Therapeutic Potential in Autoimmune Disease.

Producing Ag-specific immune responses constrained to target tissues or cells that can be engaged or disengaged at will is predicated on understanding the network of genes governing immune cell function, defining the rules underlying Ag specificity, and synthesizing the tools to engineer them. The successes and limitations of chimeric Ag receptor (CAR) T cells emphasize this goal, and advances in high-throughput sequencing, large-scale genomic screens, single-cell profiling, and genetic modification are providing the necessary data to bring it to fruition-including a broader application into the treatment of autoimmune diseases. In this review, we delve into the implementation of these developments, survey the relevant works, and propose a framework for generating the next generation of synthetic T cells informed by the principles learned from these systems approaches.

Clinical trials in multiple sclerosis: past, present, and future.

For the past four decades, multiple sclerosis (MS) has been a focus for clinical trial development and execution. Advances in translational neuroimmunology have led to the development of effective disease-modifying therapies (DMTs) that greatly benefit patients with MS and mitigate their burden of disease. These achievements also stem from continued progress made in the definition and discovery of sensitive disease diagnostic criteria, objective disability assessment scales, precise imaging techniques, and disease-specific biomarkers. As a result, our knowledge of MS pathophysiology is more mature; the established clinical practice for the diagnosis and management of MS could serve as a roadmap to guide the development of more disease-specific interventions. In this article we briefly review the main achievements in the evolution of clinical trials for MS, and discuss opportunities for improvements.

Efficacy of Disease Modifying Therapies in Progressive MS and How Immune Senescence May Explain Their Failure.

The advent of disease modifying therapies (DMT) in the past two decades has been the cornerstone of successful clinical management of multiple sclerosis (MS). Despite the great strides made in reducing the relapse frequency and occurrence of new signal changes on neuroimaging in patients with relapsing remitting MS (RRMS) by approved DMT, it has been challenging to demonstrate their effectiveness in non-active secondary progressive MS (SPMS) and primary progressive MS (PPMS) disease phenotypes. The dichotomy of DMT effectiveness between RRMS and progressive MS informs on distinct pathogeneses of the different MS phenotypes. Conversely, factors that render patients with progressive MS resistant to therapy are not understood. Thus far, age has emerged as the main correlate of the transition from RRMS to SPMS. Whether it is aging and age-related factors or the underlying immune senescence that qualitatively alter immune responses as the disease transitions to SPMS, that diminish DMT effectiveness, or both, is currently not known. Here, we will discuss the role of immune senescence on different arms of the immune system, and how it may explain relative DMT resistance.

Coevolution-based inference of amino acid interactions underlying protein function.

Protein function arises from a poorly understood pattern of energetic interactions between amino acid residues. Sequence-based strategies for deducing this pattern have been proposed, but lack of benchmark data has limited experimental verification. Here, we extend deep-mutation technologies to enable measurement of many thousands of pairwise amino acid couplings in several homologs of a protein family - a deep coupling scan (DCS). The data show that cooperative interactions between residues are loaded in a sparse, evolutionarily conserved, spatially contiguous network of amino acids. The pattern of amino acid coupling is quantitatively captured in the coevolution of amino acid positions, especially as indicated by the statistical coupling analysis (SCA), providing experimental confirmation of the key tenets of this method. This work exposes the collective nature of physical constraints on protein function and clarifies its link with sequence analysis, enabling a general practical approach for understanding the structural basis for protein function.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 ß-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to ß-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

PD-L1 regulates the development, maintenance, and function of induced regulatory T cells.

Both the programmed death (PD) 1-PD-ligand (PD-L) pathway and regulatory T (T reg) cells are instrumental to the maintenance of peripheral tolerance. We demonstrate that PD-L1 has a pivotal role in regulating induced T reg (iT reg) cell development and sustaining iT reg cell function. PD-L1(-/-) antigen-presenting cells minimally convert naive CD4 T cells to iT reg cells, showing the essential role of PD-L1 for iT reg cell induction. PD-L1-coated beads induce iT reg cells in vitro, indicating that PD-L1 itself regulates iT reg cell development. Furthermore, PD-L1 enhances and sustains Foxp3 expression and the suppressive function of iT reg cells. The obligatory role for PD-L1 in controlling iT reg cell development and function in vivo is illustrated by a marked reduction in iT reg cell conversion and rapid onset of a fatal inflammatory phenotype in PD-L1(-/-)PD-L2(-/-) Rag(-/-) recipients of naive CD4 T cells. PD-L1 iT reg cell development is mediated through the down-regulation of phospho-Akt, mTOR, S6, and ERK2 and concomitant with the up-regulation of PTEN, all key signaling molecules which are critical for iT reg cell development. Thus, PD-L1 can inhibit T cell responses by promoting both the induction and maintenance of iT reg cells. These studies define a novel mechanism for iT reg cell development and function, as well as a new strategy for controlling T reg cell plasticity.