Thereza Imanishi-Kari

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a γ2a variable region

To examine germ line genes of the heavy chain variable region (VH) that might contribute to formation of antibodies of the NPb family, we have derived cDNA clones from two hybridomas making NPb antibodies. One, B1-8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the VH region, had very different D segments and had identical J segments (J2). A set of closely related germ line VH genes was then cloned from a partial Eco RI library of C57Bl/6 DNA. By comparing the germ line VH regions to the cDNA VH regions, we identified seven potential candidates for encoding the VH regions of NPb antibodies. The seven VH regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1-8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1-8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other VH genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their VH regions from the same germ line gene. Certain of the sequenced VH genes contain crippling mutations; the repertoire of germ line VH genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hydridization.

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.

Molecular basis of a mouse strain-specific anti-hapten response

The response of C57BL/6 and BALB/c mice to immunization with proteins coupled to (4-hydroxy-3-nitrophenyl)acetyl (NP) is dominated by distinctly different sets of antibodies. The VH gene family previously shown to be involved in the C57BL/6 response has now been shown to have highly homologous counterparts in BALB/c but of five sequenced BALB/c VH regions, none appeared likely to be able to encode an NP-binding protein. The active VH region from a BALB/c hybridoma making a characteristic anti-NP antibody was recovered and sequenced and shown to be quite different from the VH gene family involved in the C57BL/6 response. Comparison of the variation of the closely related VH regions between the two mouse strains showed that there are separate types of evolutionary pressures on the framework and complementarity-determining regions. The molecular basis for strain-specific immune responses appears to be that the structural divergence of VH regions between mouse strains is great enough that different strains use different VH regions for making the predominant class of antibodies to a specific hapten.

T cell-independent somatic hypermutation in murine B cells with an immature phenotype

Somatic hypermutation contributes to the generation of antibody diversity and is strongly associated with the maturation of antigen-specific immune responses. We asked whether somatic hypermutation also plays a role in the generation of the murine immunoglobulin repertoire during B cell development. To facilitate identification of somatic mutations, we examined mouse systems in which only antibodies expressing lambda1, lambda2, and lambdax light chains can be generated. Somatic mutations were found in cells, which, by surface markers, RAG expression, and rapid turnover, had the phenotype of immature B cells. In addition, expression of AID was detected in these cells. The mutations were limited to V regions and were localized in known hotspots. Mutation frequency was not diminished in the absence of T cells. Our results support the idea that somatic hypermutation can occur in murine immature B cells and may represent a mechanism for enlarging the V gene repertoire.

Altered repertoire of endogenous immunoglobulin gene expression in transgenic mice containing a rearranged Mu heavy chain gene

C57BL/6 mice transgenic for a mu heavy chain gene, the VDJ region of which came from the BALB/c hybridoma 17.2.25, expressed high levels of antibody carrying determinants specific for the transgene (idiotypes). The individual antibodies made by hybridomas from transgenic mice, however, were generally encoded by endogenous genes; in most cases the transgene was present but not expressed. The endogenous, idiotype-positive antibodies had heavy chains that were notable for the high frequencies of JH4 (as in the transgene) and VH segments from the VH81X family (unrelated to the transgene). The expression of endogenous genes mimicking the idiotype of the transgene suggests that a rearranged gene introduced into the germ line can activate powerful cellular regulatory influences.

Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells

Transgenic expression of TLR7 results in the expansion and hyperactivation of T1 B cells in response to endogenous RNA complexes, leading to increased autoantibody production.

Dosage of X‐linked Toll‐like receptor 8 determines gender differences in the development of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease with a high incidence in females and a complex phenotype. Using 564Igi mice, a model of SLE with knock‐in genes encoding an autoreactive anti‐RNA Ab, we investigated how expression of Toll‐like receptors (TLRs) in B cells and neutrophils affects pathogenesis. We established that TLR signaling through MyD88 is necessary for disease. Autoantibody was produced in mice with single deletions of Tlr7, Tlr8, or Tlr9 or combined deletions of Tlr7 and Tlr9. Autoantibody was not produced in the combined absence of Tlr7 and Tlr8, indicating that TLR8 contributes to the break in tolerance. Furthermore, TLR8 was sufficient for the loss of B‐cell tolerance, the production of class‐switched autoantibody, heightened granulopoiesis, and increased production of type I IFN by neutrophils as well as glomerulonephritis and death. We show that dosage of X‐linked Tlr8 plays a major role in the high incidence of disease in females. In addition, we show that the negative regulation of disease by TLR9 is exerted primarily on granulopoiesis and type I IFN production by neutrophils. Collectively, we suggest that individual TLRs play unique roles in the pathogenesis of systemic lupus erythematosus, suggesting new targets for treatment.

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 VH 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 VH and VL gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1VH 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 VH DHJH 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 VH domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.

Expression of an anti-RNA autoantibody in a mouse model of SLE increases neutrophil and monocyte numbers as well as IFN-I expression.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinucleic acid autoantibodies, high levels of circulating type I interferon (IFN‐I), and an IFN‐I‐dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN‐I production is poorly understood. Here, we analyzed SLE pathogenesis in 564Igi mice, an SLE‐model strain carrying gene‐targeted heavy and light chain antibody genes encoding an anti‐RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti‐RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produced IFN‐I and exhibit increased FcγRIV expression induced via an IFN‐I autocrine loop. A direct effect of IFN‐I on 564Igi BM B cells and neutrophils was supported by their upregulation of “IFN‐I signature genes”. In addition, 564Igi developing B cells showed upregulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti‐RNA autoantibody is sufficient to induce an increase of BM, blood, and spleen IFN‐I‐producing neutrophils, and suggest a mechanism by which autoantibody and IFN‐I contribute to SLE by activating B lymphocytes, neutrophils, and monocyte effector cells in vivo.

Female bias in systemic lupus erythematosus is associated with the differential expression of X-linked toll-like receptor 8

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70–90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.