Extensive intrathecal T cell renewal following hematopoietic transplantation for multiple sclerosis.

A recent study of autologous hematopoietic stem cell transplantation (AHSCT) for active relapsing-remitting multiple sclerosis (RRMS) showed efficacy in preventing disease worsening. However, the immunologic basis for efficacy remains poorly defined. Multiple sclerosis pathology is known to be driven by inflammatory T cells that infiltrate the CNS. Therefore, we hypothesized that the preexisting T cell repertoire in the intrathecal compartment of active RRMS participants was ablated and replaced with new clones following AHSCT. T cell repertoires were assessed using high-throughput TCRß chain sequencing in paired cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T cells from participants that underwent AHSCT, before and up to 4 years following transplantation. More than 90% of the preexisting CSF repertoire in participants with active RRMS was removed following AHSCT and replaced with clonotypes predominantly generated from engrafted autologous stem cells. Of the preexisting clones in CSF, approximately 60% were also detected in blood before therapy, and concordant treatment effects were observed for clonotypes in both compartments following AHSCT. These results indicate that replacement of the preexisting TCR repertoire in active RRMS is a mechanism for AHSCT efficacy and suggest that peripheral blood could serve as a surrogate for CSF to define mechanisms associated with efficacy in future studies of AHSCT.

Cytomegalovirus Exposure in the Elderly Does Not Reduce CD8 T Cell Repertoire Diversity.

With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8+ T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR ß-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8+ T cell repertoire diversity.

A Public Database of Memory and Naive B-Cell Receptor Sequences.

The vast diversity of B-cell receptors (BCR) and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.

Dynamics of the cytotoxic T cell response to a model of acute viral infection.

UNLABELLED: A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8(+) T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8(+) T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor ß-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼ 2,000 CD8(+) T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion. IMPORTANCE: The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells.

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip.

The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.