In vivo and in vitro IgE isotype switching in human B lymphocytes: Evidence for a predominantly direct IgM to IgE class switch program

Molecular analysis of circular excision products and composite genomic switch regions has demonstrated that in mice, immunoglobulin (Ig) isotype switching from IgM to IgE often proceeds sequentially via IgG1. Based on analysis of Ig production in cell cultures, it has been suggested that human B cells may switch to IgE via IgG4, whereas limited molecular data from in vitro switched B cells suggest a direct IgM to IgE switch program. To obtain a quantitative assessment of direct versus sequential IgE switching in humans, we have analyzed the nucleotide sequences of 29 composite Sμ/S? switch regions from freshly isolated human B lymphocytes from patients with atopic dermatitis and from B lymphocytes induced to switch to IgE synthesis in vitro. The data show that in these B cells IgE isotype switching progressed directly from IgM to IgE. We conclude that, in contrast to the murine IgM/IgE switch program, the IgM to IgE switch in B lymphocytes from patients with atopic dermatitis as well as in vitro stimulated B cells from healthy donors preferentially proceeds via direct Sμ to S? switch recombination.     

A rapid method for starting a culture for the establishment of Epstein-Barr virus-transformed human lymphoblastoid cell lines

Summary We developed a simple and efficient procedure for the establishment of Epstein-Barr virus-transformed human lymphoblastoid cell lines. B-lymphocytes were obtained by centrifugation after hemolysis of red cells with a hemolysis buffer, instead of Ficoll-Parque gradient. We can start a primary culture within 15 min by using this method.     

Isolation of heavy chain class switch variants of a monoclonal anti-DC1 hybridoma cell line: effective conversion of noncytotoxic IgG1 antibodies to cytotoxic IgG2 antibodies

Spontaneously arising class switch variants of the Genox 3.53 hybridoma cell line were isolated. They secrete IgG2a or IgG2b monoclonal antibodies of anti-DC1 specificity identical to that of the IgG1 secreting parental cell. In contrast to the parental monoclonal antibody, those secreted by the variants are cytotoxic to peripheral blood B lymphocytes of DC1 positive individuals and are thus compatible with existing HLA typing techniques. This provides a general method for converting noncytotoxic anti-HLA antibodies into cytotoxic typing reagents.     

Regulation of secretion of IL-4 and IgG1 isotype by melatonin-stimulated ovalbumin-specific T cells

In the present study, we describe the potential role of melatonin, a pineal hormone, in regulating the activation of the antigen-specific T cell response. Melatonin encouraged the proliferation of Th cells and improved their ability to secrete IL-4, but down-regulated the levels of IL-2 and interferon-gamma (IFN-γ). Melatonin, however, could not exert any influence on the T cells of unprimed mice. On studying the regulation of subclass of IgG isotype, melatonin specifically enhanced the secretion of antigen-specific IgG1 antibodies and decreased the yield of IgG2a isotype. The results suggest that melatonin possibly acts by selectively activating a Th2-like immune response.     

Recombinant full-length human IgG1s targeting hormone-refractory prostate cancer

We have used a naive human single-chain fragment variable (scFv) library as a source of random shape repertoire to directly probe the altered surface chemistry of tumor cells. We reported previously the identification of more than 90 internalizing phage monoclonal antibodies targeting prostate cancer cells, including those that are hormone refractory. In this report, we describe the conversion of a panel of those scFvs into full-length human immunoglobulins (IgGs) and show that tumor specificity is retained. We have further shown that antibodies isolated from a naive phage display library can nevertheless be of high affinity towards target tumor cells. In addition, full-length IgGs retain the functionality of parental scFvs including the ability to rapidly enter target cells through receptor-mediated endocytosis and thereby to mediate efficient and specific intracellular payload delivery to tumor cells. We have used recombinant IgGs to immunoprecipitate target antigens and analyzed their molecular composition by mass spectrometry. We have identified one target antigen as activated leukocyte cell adhesion molecule (ALCAM)/MEMD/CD166 and have further studied tissue specificity of this internalizing ALCAM epitope by immunohistochemistry. Our study shows that cell type-specific internalizing human antibody can be readily identified from a naive phage antibody display library, characterized with regards to sequence, affinity, tissue specificity, and antigen identity, and modified genetically and chemically to generate various forms of targeted therapeutics.     

An effective method for establishing human B lymphoblastic cell lines using epstein-barr virus

Summary Methods are described for the efficient transformation of fresh or cryopreserved human B lymphocytes to produce continuous, lymphoblastic cell lines. Lymphocytes are separated from whole blood by centrifugation through Ficoll. They are transformed by exposure to Epstein-Barr Virus (EBV) obtained as a supernatant from ATCC. CRL 1612 (B95-8) cells. Virus production is verified in advance by immunoperoxidase staining after application of a monoclonal antibody to EBV capsid antigen [ATCC.HB 168 (72A1)]. The addition of irradiated feeder cells [ATCC.CCL 17 (MRC-5)] is important to enhance efficiency in lymphoblast culture initiation.     

GM1, a ganglioside that specifically enhances immunoglobulin production and proliferation in human plasma cells

The effects of gangliosides on human plasma cell responses were studied. Among the various gangliosides tested, only GM1 enhanced immunoglobulin (Ig) production and proliferation in the human plasma cell lines, IM-9 and AF-10, while other gangliosides (GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b) had no effect. Among the various cytokines tested, including interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, interferon (IFN)-α and IFN-γ, only IL-6 enhanced Ig production and proliferation in IM-9 and AF-10 cells. However, the enhancement of plasma cell responses by GM1 was specific and was not mediated by IL-6, since GM1 activity was blocked by anti-GM1 monoclonal antibody (mAb), but not by control IgM, anti-IL-6 Ab or the anti-IL-6 receptor mAb, PM1. Conversely, the enhancement by IL-6 was blocked by anti-IL-6 Ab and PM1, but not by anti-GM1 mAb. GM1, but not other gangliosides, also enhanced Ig production and proliferation in freshly separated plasma cells from patients with plasma cell leukemia and in plasma cells generated in vitro. These actions of GM1 were specifically blocked by anti-GM1 mAb, but not by anti-IL-6 Ab or PM1. These results indicate that GM1 may be an important regulator of plasma cell responses.     

Characterization of human IgG repertoires in an acute HIV-1 infection

All known broadly neutralizing antibodies (bnAbs) are highly somatically mutated and therefore significantly differ from their germline predecessors. Thus although the mature bnAbs bind to conserved epitopes of the HIV-1 envelope glycoprotein (Env) with high affinity their germline predecessors do not or weakly bind Envs failing to initiate an effective immune response. The identification of less somatically mutated bnAbs and/or antibody maturation intermediates that are clonally related to bnAbs may be useful to circumvent the major problem of initiating immune responses leading to elicitation of bnAbs. Here, we describe the identification of IgG antibodies from an acutely HIV-1-infected patient using a combination of phage display and high-throughput sequencing. We found two antibodies with only a single point mutation in the V region of their heavy chain variable domains compared to their putative germline predecessors which bound with high affinity to several Envs. They targeted the Env gp41 and did not neutralize HIV-1. Using high-throughput sequencing, we identified several highly abundant CDR3s, germline-like as well as somatically mutated V genes in the VH/VL repertoires of the patient which may provide antibody intermediates corresponding to known bnAbs as templates for design of novel HIV-1 vaccine immunogens.     

Immunoglobulin allotypes influence IgG antibody responses to hepatitis C virus envelope proteins E1 and E2

Immunoglobulin (Ig) GM and KM allotypes—genetic markers of γ and κ chains, respectively—are associated with the outcome of hepatitis C virus (HCV) infection, but the underlying mechanisms are not well understood. We hypothesized that GM and KM allotypes could contribute to the outcome of HCV infection by influencing the levels of IgG antibodies to the HCV glycoproteins E1E2. We serologically allotyped 100 African American individuals with persistent HCV infection for GM and KM markers and measured anti-E1E2 antibodies. Subjects with the GM 1,17 5,13 phenotype had significantly higher levels of anti-E1E2 antibodies than subjects who lacked this phenotype (p = 0.008). Likewise, subjects with the KM 1–carrying phenotypes had higher levels of anti-E1E2 antibodies than subjects who lacked these phenotypes (p = 0.041). Median titers were fourfold higher in persons expressing both GM 1,17 5,13 and KM 1–carrying phenotypes compared with those who lacked these phenotypes (p = 0.011). Interactive effects of these GM-KM phenotypes were previously found to be highly significantly associated with spontaneous HCV clearance. Results presented here show that Ig allotypes contribute to the interindividual differences in humoral immunity to the HCV epitopes, a finding that may provide a mechanistic explanation for their involvement in the outcome of HCV infection.     

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q‐mediated complement system in the basal chordate

The origin of the classical complement pathway remains open during chordate evolution. A C1q‐like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan‐binding lectin‐associated serine protease‐2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q‐like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q‐mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway.     

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12

Abstract: We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.     

IgG1 and IgG4 antibody responses to the dust mite Lepidoglyphus destructor in a naturally exposed farming population

The natural humoral immune response to the dust mite Lepidoglyphus destructor was assessed by comparing the IgG1 and IgG4 responses elicited in allergic ( n = 44) and healthy ( n = 16) individuals in a farming population chronically exposed to this allergen. With the aid of an immunoblotting technique and ELISA, the sera were analyzed for anti- L. destructor antibodies. While the majority of sera from the allergy group displayed several bands for both IgG1 and IgG4, the nonatopic healthy group was negative as analyzed in immunoblotting. When they were analyzed in ELISA, there was a significantly higher response in the allergy group than in the healthy group for IgG4, but not for IgG1. Taken together, these results imply that the immune system of individuals spared from allergic reactions to L. destructor not only lacks IgE antibodies but also seems largely to "ignore" these allergens/antigens despite exposure.