Gregory C. Ippolito

The promise and challenge of high-throughput sequencing of the antibody repertoire.

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

Developmental and species-divergent globin switching are driven by BCL11A

The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian β-globin loci have served as a model for gene regulation during development. Transgenic mice containing the human β-globin locus, consisting of the linked embryonic (ε), fetal (γ) and adult (β) genes, have been used as a system to investigate the temporal switch from fetal to adult haemoglobin, as occurs in humans. Here we show that the human γ-globin (HBG) genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human γ-globin expression identified by genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human γ-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of β-globin gene regulation in mice and humans, we have shown that alterations in the expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets—peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing

Manipulation of a transcriptional repressor promotes expression of protective fetal globin genes. Persistence of human fetal hemoglobin (HbF, α2γ2) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.

Complement activation in factor D-deficient mice

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the α chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg2+, whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.

Identification and characterization of the constituent human serum antibodies elicited by vaccination.

Significance Most vaccines confer immunity by eliciting long-term production of antibodies that bind to and neutralize the vaccine antigen. Remarkably, very little is known regarding the identities, sequence diversity, relative concentrations, or binding functionalities of the mAbs that comprise the serum repertoire elicited by vaccination. Here, we have delineated the constituent antibodies of the human serum IgG repertoire after vaccination and examined their relationship to the antibody V gene repertoire encoded by circulating B cells. The results detail the molecular composition and characteristics of the vaccine-specific serum antibody repertoire and demonstrate differences between the end-point response (the serum antibodies) and the peripheral B cells responding to the vaccine. Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT+ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd ∼ 10−8–10−10 M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3−CD14−CD19+CD27++CD38++CD20−TT+) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.

Patterns of Spinal Sensory-Motor Connectivity Prescribed by a Dorsoventral Positional Template

Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype or, indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity and thus motor coordination.

FOXP1: A potential therapeutic target in cancer

Forkhead Box P1 (FOXP1) is a member of the FOX family of transcription factors which have a broad range of functions. Foxp1 is widely expressed and has been shown to have a role in cardiac, lung and lymphocyte development. FOXP1 is targeted by recurrent chromosome translocations and its overexpression confers a poor prognosis in a number of types of lymphomas, suggesting it may function as an oncogene. In contrast, FOXP1 localises to a tumour suppressor locus at 3p14.1 and loss of FOXP1 expression in breast cancer is associated with a worse outcome, suggesting FOXP1 may function as a tumour suppressor in other tissue types. These data suggest that FOXP1 may not only be useful in prognosis but also may be used to develop FOXP1-directed therapeutic strategies.

Forced usage of positively charged amino acids in immunoglobulin CDR-H3 impairs B cell development and antibody production

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.

Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development.

Proper thymocyte development is required to establish T-cell central tolerance and to generate naive T cells, both of which are essential for T-cell homeostasis and a functional immune system. Here we demonstrate that the loss of transcription factor Foxp1 results in the abnormal development of T cells. Instead of generating naive T cells, Foxp1-deficient single-positive thymocytes acquire an activated phenotype prematurely in the thymus and lead to the generation of peripheral CD4(+) T and CD8(+) T cells that exhibit an activated phenotype and increased apoptosis and readily produce cytokines upon T-cell receptor engagement. These results identify Foxp1 as an essential transcriptional regulator for thymocyte development and the generation of quiescent naive T cells.