Preferential Damage to IgM Production by Ultraviolet B in the Cells of Patients with Bloom's Syndrome

In most patients with Bloom's syndrome (BS), selective IgM deficiency is commonly found. We examined proliferative responses by incorporation of [³H]-thymidine and the production of immunoglobulin after ultraviolet B (UVB) irradiation in the cells of two patients with BS. With regard to the proliferative responses in peripheral blood mononuclear cells (PBMC) cultured with pokeweed mitogen (PWM), the patients' PBMC were more sensitive to UVB irradiation than controls. Although the effect of UVB irradiation in lymphoblastoid cell lines (LCL) after 0 days of culture showed no difference between one patient and controls, the patient's LCL were more sensitive to UVB than the controls after 3 and 7 days of culture. These results suggest that the proliferative responses of the patient's LCL recovered later than those of controls. IgM production was the most sensitive to UVB in the patients' PBMC and LCL. IgG and IgA production in the patients' PBMC and LCL showed the same sensitivity as controls. From our results, it is suspected that the preferential damage to IgM production by UVB is connected with the selective IgM deficiency of BS.     

Evidence for a human IgG1 class switch program

In activated murine B lymphocytes, immunoglobulin class switch recombination occurs as a highly regulated process which is targeted to distinct switch regions. Here we present first evidence that in human B lymphocytes, switch recombination is targeted to distinct switch regions as well. In a panel of clonally unrelated IgG1-expressing human B cells, immortalized by Epstein-Barr virus (EBV) transformation, seven out of nine cells show switch recombination between Sμ and Sγ1 on both alleles, the active and inactive one. The remaining cells show no switch recombination on the inactive IgH locus. The very strong correlation of switch recombination on both alleles of IgG1-expressing cells proves that class switch recombination to IgG1 is not random but directed in human B lymphocytes.     

DIVERSITY IN DNA REARRANGEMENTS AND IN RNA EXPRESSIONS OF IMMUNOGLOBULIN GENE ON COMMON VARIABLE IMMUNODEFICIENCY

Six heterogeneous common variable immunodeficiency (CVID) patients were analysed for germ-line DNA, DNA rearrangements, and RNA expressions of immunoglobulin (Ig) gene by Southern or northern blotting using appropriate probes. We detected no polymorphism in neutrophil DNA hybridized to a C μ and a C γ probe. In three patients, both serum Ig and Ig-bearing cells were scarcely detected, and by northern hybridization methods, neither μ mRNA, γ mRNA, α mRNA nor k mRNA was detected. However, one Epstein-Barr virus-transformed B lymphoblastoid cell line (LCL) of these three patients was different from the germ line in the region of J_H, Cγ, and Ck , and expressed μ mRNA at a higher level. The B cell defects of these three patients lay on the B cell maturation stage similar to X-linked agammaglobulinaemia (XLA). In two others among the six CVID patients, serum IgM and IgM-bearing cells were detected to a certain degree, and by northern hybridization, μ mRNA was detected at a lower level, but neither μ mRNA, α mRNA, nor k mRNA was detected. One LCL of these two patients could express μ mRNA at the normal level. In the last patient, the serum IgM was normal, serum IgG and IgA were somewhat low, Ig-bearing cells were normal, μ mRNA and k mRNA were detected at the normal level, and γ mRNA and k mRNA were detected at a lower level. The defect of this patient affected the class switch stage. These results showed that primary B cell defects in CVID occurred at several B cell differentiation stages which could be classified by expression of the Ig gene, and at the degree of clonal diversity in the B cell repertoire. Furthermore, this study provides support for the idea that the CVID defect is related to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.     

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 syngeneic idiotype is immunogenic when borne by IgM but tolerogenic when joined to IgG

Some syngeneic monoclonal antibodies (mAb) elicit immune responses like conventional T-dependent antigens. To find out whether the heavy chain class (isotype) plays a role for the immunogenicity of an idiotype (Id), we isolated rare subclones of an IgM mAb (termed Id3) in which the variable region of the heavy chain (V_H) is associated with a new constant region (C_H). The V_H-Id3 gene is a member of the murine 36–60 family and probably has three replacement mutations. The light chain V gene is germ-line Vλ2. IgM, IgG1, IgG2a and IgG2b variants of Id3 were purified from protein-free medium and injected without adjuvant into BALB/c mice. The parental 19S IgM mAb given subcutaneously (s.c.) elicited a vigorous humoral response against Id3; in comparison, monomeric 8S IgM was a much weaker immunogen. Unlike IgM, multiple challenges with the IgG switch variants failed to induce anti-Id3 Ab. IgG variants gained immunogenicity if they were purified from medium containing fetal calf serum, mixed with complete Freund's adjuvant or injected into mice primed with IgM-Id3. Pretreatment with 100 μg s.c. + 50 μg of the IgG2a variant extinguished the Ab response to parental IgM, but the response to adjuvant-free bovine serum albumin was intact. Therefore, the tolerance induced by the IgG2a switch variant is antigen-specific and not due to toxicity. Significant inhibition of the Ab response to parental IgM was observed after treatment with 4 μg of the IgG2a switch variant. Administration of the IgG1 and IgG2b switch variants also inhibited this response significantly. Thus, the outcome of an encounter with Id3 is strongly influenced by the C_H isotype to which the Id is joined. This suggests novel ways to minimize unwanted Ab responses against Id of human therapeutic mAb. In the context of the theory of Id networks, we suggest that dominant B cell clones can preempt anti-Id Ab responses against themselves by early switching from IgM to IgG secretion, before immunogenic IgM Ab have had time to activate anti-Id B cells.     

Pokeweed mitogen-induced immunoglobulin secretory responses of thymic B cells in myasthenia gravis: selective secretion of IgG versus IgM cannot be explained by helper functions of thymic T cells

The thymus, with its striking B cell infiltrates, is widely regarded as an important element in the pathogenesis of myasthenia gravis (MG) but its role remains to be elucidated. To gain further insight into the functional properties of MG thymic B cells, we studied the heavy chain isotype of immunoglobulin they produced in vitro in response to the T cell-dependent polyclonal activator pokeweed mitogen (PWM). MG thymic cells secreted prominent amounts of IgG but little IgM. In contrast, peripheral blood mononuclear cells (PBM) of the same subjects secreted similar amounts of IgG and IgM as did PBM of control subjects. In cell admixture experiments, MG thymic T cells, like PBM T cells, helped autologous PBM B cells produce IgM as well as IgG, although the overall magnitude of help for both isotypes appeared less than that of PBM T cells. Thus, in response to PWM, MG thymic B cells are largely committed to an IgG response and this likely reflects the intrinsic properties of these cells rather than the immunoregulatory properties of thymic T cells. This IgG isotype switch likely reflects in vivo activation events.     

DNA immunization in relB-deficient mice discloses a role for dendritic cells in IgM → IgG1 switch in vivo

A single intraspleen inoculation of plasmid DNA coding for an immunoglobulin heavy chain gene initiates immunity and establishes immunologic memory against the antigenic determinants of transgenic immunoglobulins, somatic transgene immunization. During priming mice produce IgM but not IgG1 antibodies. Since IgM → IgG1 class switch occurs spontaneously during the primary immune response to protein antigens we investigated possible mechanisms for failure of spontaneous isotype switch in vivo in this model of immunity. We found that inoculation of plasmid DNA in the form of a chimeric gene coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to drive IgG1 class switch readily after priming. Since GM-CSF activates cells of the dendritic lineage we tested the possibility that dendritic cells (DC) may be involved in regulating IgM → IgG1 switch. To this end we used bone marrow chimeras constructed from mice carrying the null mutation for the relB member of the NF-κB/Rel family as these mice lack bone marrow-derived mature DC. RelB (-/-) mice and (-/-) bone marrow chimeras inoculated with DNA/GM-CSF did not produce IgG1 antibodies during the primary immune response. Since relB (-/-) bone marrow chimeras lack DC of donor origin but possess resident follicular dendritic cells we conclude that Ig class switch in vivo is regulated by the function of interdigitating dendritic cells (IDC). Thus, IDC may contribute to the qualitative aspects of the emerging immune response.     

Effects of interleukin-4 and interferon-γ on the secretion of IgG4 from human peripheral blood mononuclear cells

Whereas IgE antibodies are linked with allergy, IgG4 antibodies may reflect the state of immunity and protection against a particular antigen. It has been shown that interleukin (IL)-4 is required for induction of IgE synthesis. In order to elucidate the role of IL-4 in the production of IgG4 and to compare IgG4 and IgE regulatory processes, we quantified these immunoglobulin isotypes after in vitro culture of peripheral blood mononuclear cells (PBMC) in the presence of IL-4. The production of IgG4 was increased by IL-4 under the same conditions which are optimal for IgE production but not among PBMC from all donors, depending on the magnitude of spontaneous IgG4 secretion: IL-4 was effective only when the spontaneous secretion of IgG4 was < 7% of the total IgG secretion; it had no effect when spontaneous IgG4 production was >7% of total IgG. The IL-4-induced IgE response was consistently obtained when IgG4 was < 7% of total IgG but was markedly diminished or absent when IgG4 was > 7% of total IgG. If Staphylococcus aureus strain Cowan 1 (SAC) was present during the 48-h preincubation step, spontaneous IgG4 production was increased, but the stimulatory effect of this mitogen on immunoglobulin production, including IgG4, was markedly blocked by the addition of IL-4. In contrast, IL-4-induced IgE synthesis was strongly blocked by the presence of SAC. Finally, secretion of IgG4 (spontaneous and IL-4-induced) was suppressed among cells from most donors by interferon-γ (IFN-γ). These results suggest that IL-4 has opposite effects on in vitro IgG4 production and that the in vitro synthesis of both IgG4 and IgE appears to be regulated similarly by IL-4 and IFN-γ, whereas additional signals promote the production of one isotype in preference to the other. It is possible that activated B cells respond to IL-4 less well than do nonactivated cells.     

Restricted IgG isotype profiles in T. cruzi infected mice and Chagas'' disease patients.

IgG2 was the predominant specific antibody isotype in mice chronically infected with Y strain Trypanosoma cruzi; IgG1 and IgG3 antibodies were absent or present only at very low levels. Isotype analyses of the acute phase of infection confirmed no early production of IgG1 or IgG3 and no failure in the switch from IgM to IgG. In vivo passive transfer studies of immune serum fractions showed protection to be associated only with the IgG2 isotype. A characteristic specific anti-T. cruzi IgG isotype profile (IgG1, IgG3, greater than IgG2, IgG4) was detected in a majority (39 of 50) of sera from Chagas' disease patients.     

Molecular analysis of HIV-1 gp120 antibody response using isotype IgM and IgG phage display libraries from a long-term non-progressor HIV-1-infected individual.

To characterize the variable heavy chain (VH)3 antibody response to HIV-1 gp120, we analyzed a panel of IgM and IgG1 Fab fragments from phage display isotype libraries from a long-term, non-progressor HIV-1-infected individual. The IgM Fab antibodies isolated had low affinity for gp120, were not restricted to a particular VH3 germ-line gene, and consisted mainly of unmutated VH genes. In contrast, IgG Fab fragments were gp120 specific, with high affinity and extensive somatic mutation; all were clonally related and were derived from a single VH3 germ-line gene (DP50). One IgG Fab (S8) has DP50 VH region nucleotide substitutions identical to those of IgM Fab M025 and uses similar DH and JH segments, suggesting that S8 arose from M025 by isotype switching. In addition, somatic mutation in the IgG heavy chain third complementarity-determining region results in a 100-fold affinity increase for gp120, which correlates with a similar increase in neutralization capacity. These results imply that in vivo IgM to IgG isotype switch and affinity maturation may be important for protection and long-term survival in certain HIV-1-infected individuals.