J. Donald Capra

RAPID CLONING OF HIGH AFFINITY HUMAN MONOCLONAL ANTIBODIES AGAINST INFLUENZA VIRUS

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14–21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Human CD14 mediates recognition and phagocytosis of apoptotic cells

Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 (refs 2, 3) on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome,. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with ‘non-self’ components (LPS) and ‘self’ components (apoptotic cells) produce distinct macrophage responses.

Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen

We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

Automated amino acid sequence of small peptides utilizing Polybrene

Abstract The ability of Polybrene, a quaternary ammonlum compound, to facilitate automated sequencing of small peptides has been examined. In all cases, the inclusion of Polybrene has led to dramatically increased repetitive yields and has allowed the determination of the complete sequences of a variety of peptides of different origins and lengths. Methods for using Polybrene and for reducing Polybrene-generated artifacts are discussed.

Human Immunoglobulin Heavy-Chain Variable Region Genes: Organization, Polymorphism, and Expression

Publisher Summary The division of human immunoglobulin genes into various families was initially obvious from the protein data, although it appears that significant portions of the repertoire were missed by sequencing myeloma proteins. The reasons for this still remain clouded. However, using molecular techniques, most of the gene families have now been uncovered and their numbers, location, etc. are slowly being sorted out. A major surprise has been the significant interdigitation of the human V H genes, considering the clustering of similar V H genes in the mouse. Polymorphism has always been a hallmark of the immune system, and some of the earliest insights into the genetics of immunoglobulins were deduced from examinations of the polymorphisms of the constant region of human IgG. Polymorphism in the variable regions was expected and was initially thought to be an explanation for distinctions in immune responses among different individuals. To some extent, V H polymorphism was thought to be relevant to the notion of shared idiotypic specificities among antibodies with similar specificities in the human population. At the organizational level, it remains striking that over half of the gene segments in the V H I and V H III families appear to be pseudogenes, whereas the V H IV, V H V, and V H VI families contain so few. Too little is known about the V H II family to draw inference in this regard. The forces that lead to diversification and selection in the somatic cells are likely to be similar to those that lead to preservation of specific V H genes in the germ cells.

Mature B cells class switched to IgD are autoreactive in healthy individuals

Determination of the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that, despite being derived from healthy people, antibodies from B cells that have class switched to IgD via genetic recombination (and thus become class switched to C delta [C delta-CS] cells) are highly reactive to self antigens. Over half of the antibodies from C delta-CS B cells bind autoantigens on human epithelioma cell line 2 (HEp-2) cells or antinuclear antigens, and a quarter bind double-stranded DNA; both groups of antibodies are frequently polyreactive. Intriguingly, some C delta-CS B cells have accumulated basic residues in the antibody variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, while other C delta-CS B cells are naturally autoreactive. Though the total percentage was appreciably less than for C delta-CS cells, a surprising 31% of IgG memory cell antibodies were somewhat autoreactive, and as expected, about 24% of naive cell antibodies were autoreactive. We interpret these findings to indicate either that autoreactive B cells can be induced to class switch to IgD or that autoreactive B cells that use IgD as the B cell receptor are not effectively deleted. Determination of the mechanism by which the majority of C delta-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight into the enigmatic function of the IgD antibody.

Monoclonal antibodies to immunoglobulin G4 induce histamine release from human basophils in vitro

Monoclonal murine antibodies to human IgE, IgG1, IgG2, IgG3, IgG4, IgM, and IgA1 were prepared and their abilities to stimulate histamine release from human peripheral blood leukocytes were investigated. As expected, anti-IgE caused 8% to 76% histamine release from the leukocytes of each of 14 nonallergic donors. In one subject anti-IgE alone had no effect, but a 15% histamine release did occur after the subsequent addition of a goat anti-mouse IgG antiserum to crosslink cell-bound anti-IgE molecules. Anti-IgG4 alone caused 5% to 59% histamine release from the cells of four of 14 donors. Subsequent challenge with goat anti-mouse IgG caused an additional release of up to 15% of the basophil histamine, but only from leukocytes of donors responsive to anti-IgG4 alone. No histamine release was detected after stimulation by monoclonal antibodies to IgG1, IgG2, IgG3, IgM, or IgA1. The results of this study indicate that monoclonal antibodies to IgG4 as well as to IgE can stimulate human peripheral blood leukocytes to release histamine. These observations support the theory that antigen-IgG4 interactions can stimulate histamine release from human basophils.

Calreticulin is transcriptionally upregulated by heat shock, calcium and heavy metals

Calreticulin is a new human rheumatic disease-associated autoantigen that plays a multifaceted role in cell biology. In earlier studies, this protein was shown to share an intimate relationship with the Ro/SS-A autoantigen complex, although the nature of this association continues to be debated. Since modulation of the Ro/SS-A autoantigen in epidermal keratinocytes has been implicated in the pathogenesis of subacute cutaneous lupus erythematosus and neonatal lupus erythematosus, we have begun to examine the transcriptional regulation of calreticulin. A 504 bp calreticulin promoter fragment was subcloned into a reporter gene plasmid containing firefly luciferase. Calcium ionophore, heat shock, and heavy metals such as zinc and cadmium were consistently found to increase calreticulin transcriptional activities in A431 cells (a human epidermoid squamous carcinoma cell line) under transient transfection conditions. These studies suggest that (a) calreticulin is regulated at the transcriptional level, and (b) calreticulin, like some other LE-related autoantigens, appears to function as a heat shock/stress-response gene.

Human B cell subsets.

Publisher Summary This chapter focuses on the human b cell subsets, relates it to autoimmune disease, and discusses its functions. The chapter correlates them as the cell of origin of the B cell lymphomas and leukemias. The role of reactivated memory B cells and the role they play in the germinal center (GC) reaction as well as in the eventual production of antibody secreting cells are discussed in the chapter. B cells undergo multiple types of transition in the periphery, including developmental, activation-based, and survival based. The migration trails of antigen-responsive B cells in murine lymphoid tissue by isolating Ig transcripts are described. The recurring somatic hypermutation progressively drives the Ig repertoire of memory B cells to higher affinities and suggests that the high affinity clones do not arise during a single GC passage, but can be collected during successive recall responses. The current separation schemes related to human B cell subsets, transitions in peripheral B cell development, monoclonal antibodies from human B cell subsets, and autoimmune disease and human B cell subsets are also discussed. The chapter also presents a discussion on vaccine-induced plasma cells and recombinant antibodies. The advancement of multicolor flow cytometric analysis helps to distinguish populations based on surface markers.

Rheumatoid factor V genes from patients with rheumatoid arthritis are diverse and show evidence of an antigen-driven response

Since the discovery of autoantibodies and autoimmune disease, one of the most important challenges in the field has been to understand the nature of the genes which encode autoantibodies. Between 1930-1950 a number of antibodies to autoantigens such as thyroglobuHn, immunoglobulin and DNA-nucleoprotein were described (Mackay & Bumet 1963). Through the 1960 70s. many studies were primarily concerned with the IgG class and subclass of such autoantibodies as this was particularly relevant for understanding the effector mechanisms involved in the autoimmune process. During the same period Kunkel et al. (1963) and Oudin & Michel (1963) independently defined idiotypes. and anti-idiotypic antisera were the first and most widely used immunological tools to study the structure ofthe antigen combining site and its variable region genes. Such idiotypic studies in humans were, however, primarily restricted to rare M-components with antibody activity, most of which were not associated with autoimmune disease. Early studies with V-region family-specific antisera (Eorre et al. 1976,