Scott D. Boyd

Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

B7-1 and B7-2 Have Overlapping, Critical Roles in Immunoglobulin Class Switching and Germinal Center Formation

Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freunds adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freunds adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.

Determinants of nucleosome organization in primary human cells

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.

Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing

Massively parallel sequencing of rearranged immune receptor genes permits detection and tracking of specific immune cell populations in normal and pathological contexts. Like a reporter who serially unearths fragments of a story until a plausible picture of the latest scandal emerges, scientists have over time gathered pieces of the vast amount of information inherent in the highly recombined genes of the human immune system—probing their complexity, seeking a disease diagnosis, or hunting for evidence of remission. Back in 1987, Susumu Tonegawa won the Nobel Prize in Physiology or Medicine for discovering the genetics behind the diversity of human antibodies—a process called V-D-J recombination. Now, more than 20 years later, scientists at Stanford University and 454 Life Sciences have used powerful next-generation DNA sequencing technology to comprehensively characterize the products of V-D-J recombination in both cancer patients and healthy volunteers. Indeed, this ability to exhaustively profile the human immune response will help to untangle some of biomedicine’s most knotty problems—cancer, autoimmune disease, and vaccine development. B and T lymphocytes, cells of the adaptive immune system, build the blueprints for myriad antigen-recognizing proteins—immunoglobulins (Ig) and T cell receptors—by recombination within variable (V), diversity (D), and joining (J) gene segments to rearrange the intervening highly variable DNA sequences that can specify numerous antigen recognition domains. All of this reassortment creates a repertoire of receptors that recognizes scads of molecules from foreign invaders (antigens), a process that spurs the immune system to respond to the threat. When an immune cell sporting a particular antigen receptor finds and binds its matching antigen, the cell divides repeatedly, giving rise to many genetically identical lymphocytes that target a particular antigen for elimination. In contrast to this vibrant diversity of healthy immune systems, those of people with B lymphocyte– or T lymphocyte–based cancers (lymphomas or leukemias) generate cells that express a single dominant (clonal) receptor. In the new work, Boyd et al. performed massively parallel DNA sequencing of rearranged IgH gene loci in blood and tissue samples from cancer patients and healthy people to examine the diversity of their B cells, the immune cells that make antibodies. To this end, they amplified the rearranged IgH B cell DNA with a series of primers and the polymerase chain reaction to generate bar-coded, amplified DNA mixtures. These samples were then sequenced and the information was analyzed to determine which DNA segments had been joined to generate the blueprints for the IgH immune molecules. The experimental design used by Boyd et al. employs a high-throughput deep sequencing machine and can accommodate up to 150 samples at a time, providing an intricate snapshot of the immune repertoire. From healthy individuals, the authors were able to estimate the normal complexity of the B cell repertoire. With samples from the cancer patients, they obtained disease-specific signatures of clonal B cell proliferation events. For example, in a lymph node sample from one patient, deep sequencing detected two distinct V-D-J rearrangements. This finding indicates that there were two separate clonal B cell populations in this specimen and, therefore, two different B cell lymphomas. Such signatures could be obtained at the time of disease diagnosis and then monitored on an ongoing basis and thereby used to assess the effects of anticancer therapies that target these clonal populations or for early detection of disease relapse. Characterization of immune cell populations by deep sequencing also may illuminate fundamental aspects of infectious and autoimmune diseases as well as the body’s response to vaccination, gene and cell therapies, and other surgical procedures. The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. Here, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of bar-coded amplicons were sequenced with long-read ultradeep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or reemergence of such populations after treatment.

An intact HDM2 RING-finger domain is required for nuclear exclusion of p53

The p53 tumour-suppressor protein is negatively regulated by HDM2. Recent reports indicate that the leucine-rich nuclear-export sequence (NES) of HDM2 enables it to shuttle to the cytoplasm, and that this activity is required for degradation of p53. However, it is unclear whether HDM2 is involved in nuclear export of p53, partly because p53 has itself been shown to contain a functional NES within its tetramerization domain. Here we show that co-expression of HDM2 with green fluorescent protein (GFP)-tagged p53 causes redistribution of p53 from the nucleus to the cytoplasm of the cell. This activity is dependent on binding of p53 to HDM2, and requires an intact p53 NES, but is independent of the HDM2 NES. A mutant of the HDM2 RING-finger domain that is unable to ubiquitinate p53 does not cause relocalization of p53, indicating that ubiquitin ligation or other activities of this region of HDM2 may be necessary for its regulation of p53 localization.

Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individuals Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

Diversity and clonal selection in the human T-cell repertoire

Significance A decline in the diversity of the T-cell receptor repertoire owing to thymic involution has been implicated as causing defective immune responses in the elderly. By applying next-generation sequencing of replicate TCRB libraries from highly purified T-cell subsets, and using nonparametric statistical analysis, we obtain estimates of repertoire richness in the young adult that are higher than previously reported. Although contracting with age, the repertoire remains highly diverse. These data challenge the paradigm that thymic rejuvenation is needed to maintain diversity and prevent immune incompetence in the elderly. However, we observe an increasing inequality of clonal sizes with age even among naïve T cells. This clonal selection could result in biased and possibly autoreactive immune responses. T-cell receptor (TCR) diversity, a prerequisite for immune system recognition of the universe of foreign antigens, is generated in the first two decades of life in the thymus and then persists to an unknown extent through life via homeostatic proliferation of naïve T cells. We have used next-generation sequencing and nonparametric statistical analysis to estimate a lower bound for the total number of different TCR beta (TCRB) sequences in human repertoires. We arrived at surprisingly high minimal estimates of 100 million unique TCRB sequences in naïve CD4 and CD8 T-cell repertoires of young adults. Naïve repertoire richness modestly declined two- to fivefold in healthy elderly. Repertoire richness contraction with age was even less pronounced for memory CD4 and CD8 T cells. In contrast, age had a major impact on the inequality of clonal sizes, as estimated by a modified Gini–Simpson index clonality score. In particular, large naïve T-cell clones that were distinct from memory clones were found in the repertoires of elderly individuals, indicating uneven homeostatic proliferation without development of a memory cell phenotype. Our results suggest that a highly diverse repertoire is maintained despite thymic involution; however, peripheral fitness selection of T cells leads to repertoire perturbations that can influence the immune response in the elderly.

Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens

High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10−5, with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT—information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.

Human Responses to Influenza Vaccination Show Seroconversion Signatures and Convergent Antibody Rearrangements

B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individuals secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.