Directed Ig class switch recombination in activated murine B cells

Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination.     

Deletion of Cmu genes in mouse B lymphocytes upon stimulation with LPS.

Mouse B lymphocytes can be activated polyclonally by bacterial lipopolysaccharide (LPS) to differentiate into plasmablasts. Within several days many cells perform immunoglobulin (Ig) class switching in vitro. We have purified LPS blasts expressing IgM or only IgG3 on the cell surface and analysed the DNA of these cells by Southern hybridisation blotting to detect rearrangement or deletion of CH genes. Quantitative evaluation of the Southern blots suggests that populations of surface IgG3+ (sIgG3+) cells from 6-day and sIgM+ cells from 8-day-old cultures contain only about half as many Cmu genes as spleen cells. Cmu deletion is nearly complete in populations of sIgG3+ cells from 9-day-old cultures. Therefore, upon stimulation with LPS, within a few days Cmu is deleted in most sIgG3+ cells from both chromosomes.     

Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching

We have characterized circular DNA in mouse splenocytes treated with the mitogen lipopolysaccharide (LPS) and various cytokines, including transforming growth factor beta (TGF-beta) and interleukin 4 (IL-4). Using probes of immunoglobulin heavy chain constant genes (CH), excision products of class switch recombination were identified. The majority of the clones contained the 3' portion of the switch mu (S mu) region and the 5' portion of other switch regions. Some clones contained 3'-S gamma sequences instead of 3'-S mu. This indicates that isotype switching may occur not only from C mu, but also from one of the C gamma genes to other CH genes further down-stream. In the presence of LPS, the cytokine TGF-beta enhanced the detection of 5'-S alpha-positive clones, while the lymphokine IL-4 enhanced 5'-S gamma 1 positives. The data support the notion that TGF-beta and IL-4 can direct isotype-specific class switching.     

IL-4 induces co-expression of intrinsic membrane IgG1 and IgE by murine B cells stimulated with lipopolysaccharide

IL-4 promotes IgG1 and IgE secretion by murine B cells stimulated with bacterial LPS. We show that stimulation of unprimed resting splenic B cells with LPS and 10(4) U/ml rIL-4 results in the expression of membrane (m) IgG1 and mIgE on 40 to 50% and 15 to 25% of the total B cell population, respectively, on day 4 of culture. The possibility of a significant contribution to cell surface staining by cytophilic, secreted Ig isotypes was eliminated by either the addition of anti-Fc gamma or anti-Fc epsilon R mAb during the culture or by acid treatment before staining. A similar proportion of IgE-expressing B cells are also found, after stimulation with LPS and 10(4) U/ml IL-4, by cytoplasmic staining using fluorescence microscopy. Cell sorting analysis further indicates that B cell populations that express mIgG1 and mIgE secrete these respective Ig isotypes. In addition, such cells show striking diminution in IgM secretion compared to mIgG1- or mIgE- sorted B cells. Stimulation with LPS and IL-4 (10(4) U/ml) induces co-expression of mIgG1 and mIgE on LPS-stimulated B cells; up to 75% of mIgE+ B cells co-express mIgG1 and up to 19% of mIgG1+ B cells express mIgE. This striking co-expression of mIgG1 and mIgE is mirrored by the co-expression of mIgG1 with mIgG3 and mIgG2b by B cells stimulated with LPS and 200 U/ml IL-4. Cell sorting analysis demonstrates that the B cell population that co-expresses mIgG1 and mIgE secretes both IgG1 and IgE. However, "two-color" cytoplasmic staining fails to demonstrate any B cells that simultaneously secrete both IgG1 and IgE.     

Switch region content of hybridomas: the two spleen cell Igh loci tend to rearrange to the same isotype

We have examined the switch region content of 25 hybridomas that secret antibodies of various isotypes with specificity for phosphocholine or glycoproteins of herpes simplex virus. These Southern hybridization experiments included probes for the murine JH region as well as probes for the mu, gamma 3, gamma 1, gamma 2b, gamma 2a, and alpha switch regions. For 22 of the hybridomas, the deletion model of the heavy chain switch fits the data well--all switch regions upstream of the rearranged (and expressed) switch regions are deleted and all switch regions downstream remain in the germline configuration. As exceptions to a simple deletion model of the switch recombination, we have observed two, and perhaps three, examples of switch region rearrangements downstream of an expressed heavy chain gene. The 25 hybridoma DNA samples include 28 rearranged gamma switch regions; the sizes of at least 25 of these rearranged fragments are consistent with recombination in the tandemly repeated sequences associated with gamma genes. For those hybridomas with two spleen cell-derived Igh loci, including three mu-expressers, three gamma 3-expressers, four gamma 1-expressers, and one gamma 2b-expresser, the two loci tend to be rearranged to the same switch region, suggesting that the heavy chain switch rearrangement is an isotype-specific event. The exceptions within this group include three hybridomas in which the switch seems to be incomplete--on one chromosome the JH complex is rearranged to the S gamma 3 region, while on the other it remains associated with the S mu region. A second group of hybridomas, which includes four gamma 3-expressers, have both gamma 3 and gamma 1 switch rearrangements. Each of these four hybridomas includes three rearranged JH segments, suggesting that they may be the result of an unusual differentiative pathway or a technical artifact. These experiments suggest that the heavy chain switch rearrangement in normal spleen cells is a deletion event that occurs within tandemly repeated elements. The rearrangement is mediated by factors with partial, or perhaps complete, isotype specificity.     

Differential regulation of murine B cell Fc gamma RII expression by CD4+ T helper subsets

The murine B cell FcR for IgG (Fc gamma RII) is a membrane glycoprotein reported to mediate inhibition of B cell activation and differentiation. We show that IL-4 inhibits the enhanced expression of Fc gamma RII by LPS-stimulated B cells. This activity is completely reversed by anti-IL-4 mAb and is specific, in that multiple other lymphokines tested do not exert a similar effect. This effect of IL-4 is apparent by day 1 of culture, although maximal inhibition occurs on day 4 at a concentration of 500 U/ml. The IL-4-induced inhibition of enhanced Fc gamma RII expression by LPS stimulation observed on day 4 of culture is associated with a significant reduction in the steady state level of Fc gamma RII beta gene-specific mRNA. IFN-gamma which inhibits many of the effects of IL-4 on B cells, does not reverse the IL-4-induced inhibition of Fc gamma RII membrane expression nor the levels of beta gene-specific mRNA. Fc gamma RII expression is significantly increased in B cells stimulated with antigen-specific, CD4+ T cell clones of the Th1 type (i.e., IL-2 and IFN-gamma-producing). By contrast, three different Th2 clones (i.e., IL-4-producing) fail to stimulate an increase in Fc gamma RII levels. Anti-IL-4 mAb significantly enhanced Fc gamma RII expression by Th2-stimulated B cells indicating that IL-4 was the active, inhibitory, substance produced by the Th2 cells. Supernatants from stimulated Th2 clones inhibited the enhanced expression of Fc gamma RII by LPS-stimulated B cells and this activity was completely reversed by anti-IL-4 mAb. By contrast, supernatants from stimulated Th1 clones further enhanced Fc gamma RII expression by LPS-stimulated B cells. The differential regulation of B cell Fc gamma RII expression by Th subsets may play an important role in the regulation of humoral immunity by altering the sensitivity of B cells to IgG immune complex-mediated inhibition of B cell activation and differentiation in vivo.     

Recombination events near the immunoglobulin Cmu gene join variable and constant region genes, switch heavy-chain expression, or inactivate the locus

Immunoglobulin heavy-chain expression is initiated by recombination between a variable region (VH) gene and one of several joining region (JH) genes located near the mu constant region (Cmu) gene, and the active VH gene can subsequently switch to another CH gene. That the general mechanism for CH switching involves recombination between sites within the JH-Cmu intervening sequence and the 5' flanking region of another CH gene is supported here by Southern blot hybridization analysis of eight IgG- and IgA-secreting plasmacytomas. An alternative model requiring successive VH linkage to similar JH clusters near each CH gene is shown to be very unlikely since the mouse genome appears to contain only one complement of the JH locus and no JH gene was detectable within large cloned sequences flanking germline C gamma 3 and C gamma 1 genes. Thus, VH-JH joining and CH switching are mediated by separate regions of "the joining-switch" or J-S element. In each plasmacytoma examined, the J-S element had undergone recombination within both the JH locus and the switch region and was shown to be linked to the functional CH gene in an IgG3, and IgG1, and three IgA secretors. Both JH joining and CH switching occurred by deletion of DNA. Switch recombination occurred at more than one site within the J-S element in different lines, even for recombination with the same CH gene. Significantly, although heavy-chain expression is restricted to one allele ("allelic exclusion"), all rearranged in each plasmacytoma. Some rearrangements were aberrant, involving, for example, deletion of all JH genes from the allele. Hence, an error-prone recombination machinery may account for allelic exclusion in many plasmacytomas.     

Isotype switching in human B lymphocyte malignancies occurs by DNA deletion: evidence for nonspecific switch recombination

The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.     

Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells.

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a substrate for detecting the presence of immunoglobulin heavy chain constant region (CH) gene switch (S) recombination activity in murine pre-B cells. ZN(Smu/S gamma 2b)tk1 contains a neomycin (neo) resistance gene in addition to the herpes simplex virus thymidine kinase (Htk) gene which is positioned between murine Smu and S gamma 2b sequences. Stable acquisition of the ZN(Smu/S gamma 2b)tk1 vector was selected in G-418 and switch region recombination within these proviruses was selected by resistance to the drug bromodeoxyuridine (BUdR). Fluctuation analyses of ZN(Smu/S gamma 2b)tk1 infected 18-8tk- and 38B9tk- pre-B lines revealed Htk gene inactivations with apparent frequencies of 5 X 10(-5) and 1 X 10(-5) events/cell/generation, respectively, while G-418 resistant Ltk- fibroblasts lost the HTK phenotype at an apparent rate of 4 X 10(-8). Southern blot analysis demonstrated that switch recombination caused the deletion of the Htk gene in all pre-B clones examined while the loss of Htk in Ltk- clones was not mediated by S region recombination. In 21 out of 24 pre-B clones, the recombinations involved the tandemly repetitive portions of the Smu and S gamma 2b sequences. These results demonstrate that the CH gene S region segments inserted into ZN(Smu/S gamma 2b)tk1 are sufficient for B-cell-specific recombination/deletion within the S region tandem repeats.