The rearranged V(H) domain of a physiologically selected anti-single-stranded DNA antibody as a precursor for formation of IgM and IgG antibodies to diverse antigens.

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.

Adult bone marrow contains precursors for CD5+ B cells.

To determine directly whether B cell precursors of adult origin are capable of generating CD5+ B cells, we reconstituted neonatal C3H. SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5+. This confirms that in vivo generated CD5+ B cells can be adult derived. After anti-IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow-reconstituted mice expressed surface CD5. Sequence analysis of expressed VHDJH genes from the CD5+ B cells present after anti-IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5+ B cells of adult origin rather than from long-lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5+ B cells and that unlike fetal liver-derived precursors these express terminal deoxynucleotidyl transferase.

Endogenous Ig production in mu transgenic mice. I. Allelic exclusion at the level of expression.

Our findings indicate that allelic exclusion is maintained in B cells that successfully rearrange and express and endogenous H chain gene, despite carrying a functionally rearranged H chain transgene (17.2.25 mu a). Cloned hybridomas having a functionally rearranged endogenous H chain gene as well as the transgene produce only endogenous gene products. Some of these hybridoma cultures, upon continuous growth, will secrete transgene as well as endogenous gene products. However, the two H chain polypeptides appear to be made by different cells. In each of three cases of such "double producer" lines examined, further subcloning at this time reveals two types of clones: those that secrete only transgene and those that secrete only endogenous H chains. The clones producing transgene product have lost the functionally rearranged endogenous H chain genes, whereas the clones producing endogenous gene products still contain both transgene and endogenous functionally rearranged H chain genes. These results indicate that hybridomas expressing endogenous Ig have transgene copies that are potentially functional but are reversibly silenced by an unknown mechanism and suggest that inhibition of transgene expression may be mediated by endogenous Ig gene expression.

Endogenous Ig production in mu transgenic mice. II. Anti-Ig reactivity and apparent double allotype expression.

In 17.2.25 mu transgenic mice (M54, M95), many of the expressed Ig, whether encoded by the transgene or endogenous H chain genes, react with Ig. IgM antibodies encoded by the 17.2.25 mu transgene transfected into J558L myeloma cells are also Ig reactive. In addition, anti-Ig reactivity was manifested by antibodies of the IgM, IgG, and IgA isotypes from the transgenic mice, suggesting that this characteristic reactivity is associated with VH and VL domains of these antibodies. These antibodies bind the (Fab')2 fragment of mouse IgG1 mAb known to be directed against C mu allotypic determinants. This finding could account for the so called "double producer" B cells found in mu transgenic mice and previously identified in serologic assays conducted with two different anti-mu allotypic reagents. In transgenic mice, a high frequency of the antibodies encoded by the transgene or endogenous H chain genes react with polyclonal and monoclonal antiidiotypic antibodies raised against the 17.2.25 Id. The idiotypic and/or antiidiotypic reactivity displayed by antibodies derived from these transgenic mice is similar to that of antibodies expressed by neonatal B cells of normal mice. Thus, our data suggest that transgene expression can play an important role in shaping the endogenous repertoire of antibody specificities.

Production of 17.2.25 mu transgenic and endogenous immunoglobulin in X-linked immune deficient mice.

In M54 mice transgenic for a completely rearranged mu(a) heavy chain there is a decrease in total B cells and the rearrangement of endogenous immunoglobulin genes is partially inhibited. Surprisingly, however, endogenous immunoglobulin gene rearrangement and significant heavy chain polypeptide production does occur. We tested the hypothesis that only CD5+ B cells produce endogenous immunoglobulin by taking advantage of the fact that X-linked immune deficient (xid) mice normally are deficient in CD5+ B cells. We found that the frequency of CD5+ splenic B cells was similar in XxidY transgenic and non-transgenic F1 males, and in XxidX transgenic and non-transgenic F1 females. In both XxidX and XxidY transgenic F1 mice some, but not all, splenic B cells are CD11b+. There was a striking deficit of splenic B cells expressing endogenous immunoglobulin in XxidY transgenic mice, although this was not true for peritoneal cells. Thus, the introduction of the 17.2.25 mu transgene does not prevent the development of CD5- B cells nor does it limit endogenous immunoglobulin gene arrangement and expression solely to CD5+ B cells. However, in mice capable of expressing B cell surface CD5 or CD11 this transgene can lead to expansion of the fraction of B cells positive for these molecules. We conclude that while the introduction of the 17.2.25 mu transgene alters the frequencies of B cell populations, maturation is not limited to one subpopulation.

The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.

Endogenous immunoglobulin expression in mu transgenic mice.

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes.

Genetic basis for altered idiotype expression in the hyperimmune response to (4-hydroxy-3-nitrophenyl)acetyl hapten.

To assess the significance of somatic point mutation in the hyperimmune response to the hapten NP, an in vivo enrichment procedure was followed. Mice that expressed high titers of B1-8 idiotopic determinants were selected as donors for serial transfer of small numbers of immune spleen cells into syngeneic irradiated recipient mice. Cells expressing B1-8 idiotopic determinants were chosen for enrichment because B1-8 cross-reactive determinants constitute a significant portion of the primary response. Furthermore, B1-8 is a monoclonal antibody derived from a primary response to NP, and its heavy and light chains are unmutated products of the germ-line genes VH186.2 and VL lambda 1, respectively. The germ-line sequence is thus available for comparison with the somatic mutants that arise during enrichment and hyperimmunization. The data show that serial transfer of spleen cells from mice with a high titer of idiotypic determinants results in a dramatic decrease in the titers of antibodies that bind antigen. Three lines of evidence indicate that progeny cells from the initial lambda-positive, idiotype-bearing, antigen-binding cells are successfully transferred and expanded during successive adoptive transfers. First, the proportion of lambda-bearing antibodies relative to NP-specific lambda-bearing antibodies increases with transfer, which is consistent with mutation away from antigen binding. Second, analysis of serum antibodies and hybridoma proteins derived from transfer-recipient mice confirm the presence of idiotype-positive antibodies that do not bind antigen. Third, RNA dot blot analysis of hybridomas constructed from a recipient mouse in the fourth transfer indicates a high frequency of expression of the VH gene predominantly used in the NP response. Many of the antibodies expressed by these hybridomas not only do not bind antigen, but have also lost the determinants recognized by the anti-idiotypic reagents. Most of these VH-positive hybridomas express lambda L chain. The most likely interpretation of the data is that somatic mutation is occurring during the hyperimmune response. Because we selected donor mice that expressed a high titer of idiotype-positive, antigen-specific antibody and immunized the recipient mice, we expected to observe a selective expansion of somatic variants that bound antigen. This was not the case. The observed loss of antigen binding suggests that the majority of mutations arising result in antibodies with lower affinity for the immunizing antigen.

A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes.

Transgenic mice containing a microinjected rearranged immunoglobulin (Ig) mu heavy chain gene were examined for the effects on DNA rearrangement of the endogenous Ig genes. Abelson murine leukemia virus (A-MuLV) cell lines were isolated from pre-B cells of transgenic mice and of normal littermates. Microinjected mu gene RNA and a mu heavy chain protein were synthesized in every transgenic A-MuLV cell line. Only 10% of normal mouse A-MuLV transformants synthesized mu protein. A germ-line JH allele was observed in 40% of the transgenic lines, demonstrating that the block to endogenous Ig DNA rearrangement occurred at the first step of heavy chain DNA joining. All alleles were rearranged in normal mouse A-MuLV lines. Germline JH alleles were also detected in 10% of the transgenic hybridomas derived from proliferating B cells. Our results support a model of active prevention of rearrangement by the product of successfully rearranged mu genes.

Restricted association of V and J-C gene segments for mouse lambda chains.

The frequencies of diverse rearrangements of variable (V)lambda to joining (J)lambda gene segments were examined by Southern blot hybridization in 30 murine B-cell lines, each producing an immunoglobulin lambda light chain of known subtype (lambda 1, lambda 2, or lambda 3). For 11 out of 12 lambda 1 chains, the rearrangement was V lambda 1----J lambda 1; for 9 out of 9 lambda 2 chains, it was V lambda 2----J lambda 2; and for 8 out of 9 lambda 3 chains, it was V lambda 1----J lambda 3. Similar results were obtained by considering the partial or complete sequences at the amino acid or cDNA level of 44 other lambda chains (24 previously described): for 43 of these chains the rearranged V-J gene segments were evidently V lambda 1-J lambda 1 for 28 lambda 1 chains, V lambda 2-J lambda 2 for 10 lambda 2 chains, and V lambda 1-J lambda 3 for 5 lambda 3 chains. Of the combined total of 74 chains there were 3 with unusual V lambda rearrangements, all involving the V lambda 2 gene segment: for 2 of these unusual chains, the encoding segments were V lambda 2-J lambda 1-C lambda 1 and for one they were V lambda 2-J lambda 3-C lambda 3. Thus, the results for all 74 lambda chains show that, in contrast to the apparently unrestricted V kappa----J kappa rearrangements for kappa chains, for each of the 3 murine lambda-chain subtypes V-J recombination is severely restricted: the V lambda gene segment expressed in lambda 1 and lambda 3 chains was nearly always V lambda 1 (95% and 93%, respectively), whereas in lambda 2 chains it was without exception V lambda 2 (19 out of 19 chains). Therefore V lambda-J lambda combinatorial variation is not a significant source of amino acid sequence diversity of lambda chains of inbred mice. If the order of the lambda gene segments is 5' V lambda 2-J lambda 2C lambda 2J lambda 4C lambda 4-V lambda 1-J lambda 3C lambda 3J lambda 1C lambda 1 3', as suggested previously and by the present findings, it appears that (i) when a V lambda gene segment rearranges in a developing B cell it ordinarily recombines with a J lambda gene segment in the nearest downstream (3') cluster of J lambda C lambda segments, and (ii) V lambda rearrangement to the upstream (5') cluster is very rare and possibly may not take place at all.

Somatic variants of murine immunoglobulin lambda light chains.

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.

Characterization of the NPa idiotype through the analysis of monoclonal BALB/c anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies.

Spleen cells from BALB/c mice were fused with the nonsecreting myeloma line X63.Ag8.6.5.3 seven days after immunization with NP-CG (4-hydroxy-3-nitrophenyl) acetyl-chicken gamma-globulin. The hybrid cell lines obtained were analyzed for heavy and light chain distribution, fine specificity, and idiotype. The majority of monoclonal antibodies possessed either gamma 1 or mu chains. The distribution of L chains among these antibodies was approximately half lambda and half kappa . Thirteen monoclonal antibodies were grown as ascites tumors in mice. Examination of their fine specificity patterns showed that all of the lambda antibodies are heteroclitic and have similar fine specificity patterns. Five of the seven kappa antibodies are also heteroclitic, but their fine specificity patterns are more heterogeneous than those of the lambda antibodies. Polyspecific anti-idiotypic sera directed against pooled primary serum antibody (R a-NPa) or against individual monoclonals were used for idiotypic characterization of the monoclonal antibodies. The Ra-NP bound all of the lambda antibodies but none of the kappa antibodies suggesting that the kappa antibodies may be much more heterogeneous and were therefore not recognized in the presence of the more homogeneous lambda antibodies. Further idiotypic analysis demonstrated that the lambda antibodies, although no two are identical, are a very homogeneous group of antibodies which cross-react with one another but not with the kappa antibodies. Some, but not all, of the kappa antibodies cross-react with each other although none are cross-reactive with the lambda antibodies. Because the lambda-associated idiotype is recognized by the R a-NPa and its characteristics are similar to that of the C57BL/6 major idiotype (NPb), it is referred to as NPa. There may be a second major idiotype associated with at least some of the kappa antibodies.