Models for the dynamics and order of immunoglobulin isotype switching

Experiments show that class switch recombination (CSR) depends on the number of divisions that the cell has performed rather than on the time since stimulation. Using computer simulations of CSR dynamics in B cell populations, we addressed the following questions. How does the probability of CSR depend on the number of divisions that a cell has performed? How does the cell decide which isotype to switch to? Does this decision depend on the distance between the genes of the pre-switch and the post-switch isotype? Our results indicate that post-switch isotype choice may be determined indirectly by the probabilities of division (which is fixed) and of switching per division (which increases as a function of the number of divisions that a cell performs), and more directly by a bias in the choice of the post-switch C gene segment towards those proximal to the pre-switch C gene.     

Analysis of immunoglobulin Sgamma3 recombination breakpoints by PCR: implications for the mechanism of isotype switching.

The molecular mechanism of immunoglobulin switch recombination is poorly understood. Switch recombination occurs between pairs of switch regions located upstream of the constant heavy chain genes. Previously we showed that switch recombination breakpoints cluster to a defined subregion in the Sgamma3, Sgamma1 and Sgamma2b tandem repeats. We have developed a strategy for direct amplification of Smu/Sgamma3 composite fragments as well as Smu and Sgamma3 regions by PCR. This assay has been used to analyze the organization of Smu, Sgamma3 and a series of Smu/Sgamma3 recombination breakpoints from hybridomas and normal mitogen-activated splenic B cells. DNA sequence analysis of the switch fragments showed direct joining of Smu and Sgamma3 without deletions or duplications. Mutations were found in two switch junctions on both sides of the crossover point, suggesting that template switching is the most likely model for the mechanism of switch recombination. Statistical analysis of the positions of the recombination breakpoints in the Sgamma3 tandem repeat indicates the presence of two sub-clusters, suggesting non-random usage of DNA substrate in the recombination reaction.     

Lymphokines regulate immunoglobulin isotype expression in an antigen-specific immune response

The control of immunoglobulin class switching appears to involve T cell-derived lymphokines. Such lymphokines have been shown to affect isotype expression in polyclonally activated B cells. We show in this paper that the same lymphokines similarly control isotype expression in an antigen-specific response acting in concert with a "T cell independent" antigen. In this situation, B cell growth factor II (BCGF II) enhances the production of antigen-specific IgM antibodies, whereas the production of antigen-specific IgG1 antibodies is only observed in the presence of B cell differentiation factor gamma (BCDF gamma). These results suggest that these lymphokines (and perhaps additional ones) are involved in the control of isotype expression in antigen-specific responses.     

Expression of an unidentified immunoglobulin isotype on rabbit Ig-bearing lymphocytes.

A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.     

Homologous chromosome recombination generating immunoglobulin allotype and isotype switch variants.

We investigated whether spontaneous isotype switching in monoclonal antibody-producing hybridomas always occurs with genes on the same chromosome. Spleen cells of (BAB/ 25 X AKR/J) F1 mice, immunized with dansyl-keyhole limpet hemocyanin (DNS-KLH), were hybridized with NS-1 to generate hybridomas producing monoclonal anti-DNS antibodies of either the b or d haplotype of the BAB/25 or AKR/J parent, respectively. We selected isotype switch variants of such hybridomas using the fluorescence-activated cell sorter (FACS). Although in most cases the allotypic haplotype expressed by the parent and switch-variant hybridomas are the same, in one family of variants we noted a switch in haplotype along with the switch in isotype. This was noted in the selection of IgG2a switch variants from an IgG1 switch variant originally derived from an IgG3-producing parent. Biochemical and molecular studies confirm that the allotype switch variant expresses the same heavy-chain variable region gene complex as its parent hybridomas. As such, the allotype switch represents an example of spontaneous mitotic recombination between immunoglobulin heavy-chain genes, generating a single actively transcribed gene from loci previously positioned on different chromosomes.     

Intraclonal diversification in immunoglobulin isotype secretion: an analysis of switch probabilities

The production of all immunoglobulin isotypes except IgD was studied in a large number of single lipopolysaccharide (LPS)-reactive B cell clones. The majority, but not all, of the IgM-producing clones were found to secrete one or more other isotypes. IgG3 and IgG2b were most frequently found while IgA secretion was extremely rare. Many clones produced all four IgG subclasses and the statistical analysis of the data indicates, with a high degree of significance, that single clonal precursors give rise to progenies producing multiple isotypes. By assuming that intraclonal diversification follows the C-gene order in chromosome 12, the absolute switch probabilities of normal, unprimed LPS-reactive B cells can be calculated. The multi-potentiality of C-gene expression was further analyzed at the single cell level: a sizeable fraction of all activated B cells express two different IgG isotypes in the membrane-bound form, indicating consecutive switch events. In contrast, the majority of IgE and IgA secreting cells appear to switch directly from IgM. These results might reflect the functional relevance of S-region homologies in the control of C-gene expression.     

Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice

BACKGROUND: We have previously proposed that deamination of cytosine to uracil at sites within the immunoglobulin loci by activation-induced deaminase (AID) triggers antibody diversification. The pattern of diversification (phase 1 or 2 hypermutation, gene conversion, or switch recombination) is viewed as depending on the mode of resolution of the dU/dG lesion. A major resolution mode involves excising the uracil, an activity that at least four different enzymes can accomplish in the mouse. RESULTS: Deficiency in UNG uracil-DNA glycosylase alone is sufficient to distort the pathway of hypermutation in mice. In ung(-/-) animals, mutations at dC/dG pairs are dramatically shifted toward transitions (95%), indicating that the generation of abasic sites (which can induce transversions) has been inhibited. The pattern of substitutions at dA/dT pairs is unaffected. Class-switch recombination is substantially, but not totally, inhibited. CONCLUSIONS: The results provide strong support for the DNA deamination model for antibody diversification with respect to class-switching as well as hypermutation and, in the context of this model, suggest that (i) UNG is the major mouse DNA glycosylase responsible for processing the programmed dU/dG lesions within the immunoglobulin locus; (ii) the second (dA/dT-biased) phase of mutation is probably triggered by recognition of the initiating dU/dG lesion; and (iii) switch recombination largely proceeds via formation of an abasic site, although (iv) an UNG-independent pathway of switch recombination exists, which could reflect action by another uracil-DNA glycosylase but might alternatively be explained by a distinct pathway of resolution, for example, one involving MSH2/MSH6 recognition of the dU/dG lesion.     

Transforming growth factor beta 1 increases IgA isotype switching at the clonal level

Transforming growth factor beta 1 (TGF beta 1) has important effects on expression of the IgA isotype. TGF beta 1 alone, or in combination with IL-5 or IL-2 increases IgA secretion by populations of LPS-activated surface IgA negative (sIgA-) spleen B cells, while concurrently decreasing IgM and IgG secretion. The present study demonstrates the activity of TGF beta 1 as an IgA isotype switch factor at the clonal level. Stimulation of LPS-activated sIgA- spleen B cell populations with TGF beta 1, or a combination of TGF beta 1 and IL-2, resulted in a significant increase in total numbers of IgA secreting cells, and this increase ultimately was paralleled by an increase in total IgA secretion. Using limiting dilution analysis, TGF beta 1 was shown to increase the frequency of IgA secreting B cell clones, by approximately 20-fold. This was not accompanied by increased numbers of IgA secreting cells/clone. In contrast, IL-2 does not have activity as an IgA switch factor, but does increase IgA production by B cells already committed to secrete that isotype. Cell cycle inhibitors such as thymidine and hydroxyurea also selectively increased numbers of IgA secreting cells and total IgA secretion among populations of LPS-activated sIgA- spleen B cells. This suggests the IgA enhancing activity of TGF beta 1 may, in part, be related to its ability to inhibit cell growth.     

Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

Beswitched. The looping out model for immunoglobulin class switching

During the switch in expression of an immunoglobulin class, the gene segment encoding the constant region of the heavy chain is replaced in a way that leads to a deletion. Three different models of how this deletion is generated have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, the products of the looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch.     

Boophilus microplus: the pattern of bovine immunoglobulin isotype responses to high and low tick infestations

Cattle present variable levels of resistance to ticks and the immune correlates of these heritable phenotypes must be known in order to develop effective vaccines. The antibody responses to tick salivary antigens were examined in cattle of tick-susceptible (Holstein) and tick-resistant (Nelore) breeds. After heavy infestations, levels of IgG1 and IgG2 antibodies decreased in Holsteins and remained the same in Nelores. Conversely, levels of IgE antibodies increased in Holsteins. Different sizes of tick burdens modulated the IgG1 antibody response in a susceptible breed (Aberdeen): levels were higher than in controls in heavily infested animals, but not in those undergoing intermediary or minimal infestations. The three experimental groups presented similar levels of IgG2 antibodies. Levels of IgE antibodies were higher only in animals undergoing intermediate infestations. These results indicate that tick infestations suppress the IgG antibody response in susceptible breeds, that IgE antibodies are not protective, and that the dose of tick saliva modulates the isotype of host antibody responses.