Matthias Wabl

A quasi-monoclonal mouse.

As a model for studying the generation of antibody diversity, a gene-targeted mouse was produced that is hemizygous for a rearranged V(D)J segment at the immunoglobulin (Ig) heavy chain locus, the other allele being nonfunctional. The mouse also has no functional kappa light chain allele. The heavy chain, when paired with any lambda light chain, is specific for the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). The primary repertoire of this quasi-monoclonal mouse is monospecific, but somatic hypermutation and secondary rearrangements change the specificity of 20 percent of the antigen receptors on B cells. The serum concentrations of the Ig isotypes are similar to those in nontransgenic littermates, but less than half of the serum IgM binds to NP, and none of the other isotypes do. Thus, neither network interactions nor random activation of a small fraction of the B cell population can account for serum Ig concentrations.

Dendritic cells associated with plasmablast survival

A subset of myeloid dendritic cells is described which is associated with the ability of splenic and lymph node plasmablasts to survive and differentiate into plasma cells. Plasmablast‐associated dendritic cells (PDC) are CD11chigh, DEC‐205– and unlike conventional dendritic cells do not associate with T cells. The following findings suggest a requirement for PDC if plasmablasts are to differentiate to plasma cells. First, when large numbers of B cells are recruited into antibody responses and plasmablasts outgrow the PDC stroma, only those associated with PDC survive and differentiate into plasma cells. Conversely, if the number of PDC is increased by ligating their CD40, more plasmablasts survive on the expanded PDC stroma and differentiate into plasma cells. Finally, in T cell‐deficient mice, the plasma cells that develop atypically in the T zones in response to thymus‐independent antigens are associated with ectopic PDC.

Sequential class switching is required for the generation of high affinity IgE antibodies

Generation of anaphylaxis-inducing high affinity IgE requires sequential class switching.

Pvt1-encoded microRNAs in oncogenesis

BackgroundThe functional significance of the Pvt1 locus in the oncogenesis of Burkitts lymphoma and plasmacytomas has remained a puzzle. In these tumors, Pvt1 is the site of reciprocal translocations to immunoglobulin loci. Although the locus encodes a number of alternative transcripts, no protein or regulatory RNA products were found. The recent identification of non-coding microRNAs encoded within the PVT1 region has suggested a regulatory role for this locus.ResultsThe mouse Pvt1 locus encodes several microRNAs. In mouse T cell lymphomas induced by retroviral insertions into the locus, the Pvt1 transcripts, and at least one of their microRNA products, mmu-miR-1204 are overexpressed. Whereas up to seven co-mutations can be found in a single tumor, in over 2,000 tumors none had insertions into both the Myc and Pvt1 loci.ConclusionJudging from the large number of integrations into the Pvt1 locus – more than in the nearby Myc locus – Pvt1 and the microRNAs encoded by it are as important as Myc in T lymphomagenesis, and, presumably, in T cell activation. An analysis of the co-mutations in the lymphomas likely place Pvt1 and Myc into the same pathway.

Activation of an oncogenic microRNA cistron by provirus integration

Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs. Our results demonstrate that retroviral mutagenesis can be a potent tool for miRNA discovery.

An autoimmune disease prevented by anti- retroviral drugs

BackgroundBoth Aicardi-Goutières syndrome, a Mendelian mimic of congenital infection, and the autoimmune disease systemic lupus erythematosus can result from mutations in the gene encoding the enzyme Trex1. In mice, the absence of Trex1 causes severe myocarditis. The enzyme is thought to degrade endogenous retroelements, thus linking them to autoimmune disease. However, inhibition of reverse transcription by the inhibitor zidovudine (AZT) did not ameliorate the disease, weakening the link to retroelements.FindingsHere, we show that two other FDA-approved drugs that inhibit reverse transcriptase can ameliorate the myocarditis in Trex1-null mouse.ConclusionsThe result suggests that retroelements contribute to this hereditary form of autoimmunity, and that treatment with retroelement inhibitors might ameliorate Aicardi-Goutières syndrome in humans.

Immunoglobulin mRNA stability varies during B lymphocyte differentiation

During differentiation of B lymphocytes, the change in the amount of immunoglobulin heavy chain produced is reflected by a change in the steady state level of heavy chain mRNA. At the pre‐B cell stage, the earliest stage at which immunoglobulin chain is produced, and later at the small resting B cell stage, there is a low steady state level of heavy chain mRNA. After the small B cell has differentiated to become a plasma cell, the steady state level of heavy chain mRNA is much higher. We confirm that the transcription rate at the immunoglobulin mu heavy chain gene does not change during differentiation from the pre‐B cell to the plasma cell stage. In contrast, we show here that differences in the stability of mu mRNA are sufficient to account for the differences in the steady state level at the various differentiation stages.

Retroviral activation of the mir-106a microRNA cistron in T lymphoma

Retroviral insertion into a host genome is a powerful tool not only for the discovery of cancer genes, but also for the discovery of potential oncogenic noncoding RNAs. In a large-scale mouse T lymphocyte tumor screen we found a high density of integrations upstream of the mir-106a microRNA cistron. In tumors containing an integration, the primary transcript encoding the mir-106a cistron was overexpressed five to 20-fold compared with that of control tumors; concomitantly, the mature mir-106a and mir-363 microRNAs were highly overexpressed as well. These findings suggest the mir-106a cistron plays an important role in T cell tumorigenesis.

Expression of Aire and the Early Wave of Apoptosis in Spermatogenesis

Expression of the autoimmune regulator (Aire) protein in mice and humans is thought to be restricted to the medullary epithelial and monocyte-dendritic cells of the thymus. There it mediates expression and presentation of a large variety of proteins, including those that are peripheral organ-specific and are not expressed by other thymocytes. In this way, self-reactive T lymphocytes that would attack peripheral cells producing these proteins are confronted with the self-Ags and, as a consequence, are deleted. In this study, we show that Aire mRNA is also expressed in the testis—another tissue with promiscuous gene expression. Aire protein, however, is expressed only sporadically in spermatogonia and spermatocytes. Transcription of genes that are under Aire control in the thymus is unaffected by Aire in the testis. However, in mice with a disrupted Aire gene, the scheduled apoptotic wave of germ cells, which is necessary for normal mature spermatogenesis, is reduced, and sporadic apoptosis in adults is increased. Because Rag-1 deficiency does not abolish the effect, the adaptive immune system is not involved. We suggest that there is a link between the scheduled and sporadic apoptotic processes and propose that scheduled apoptosis provides a counterselection mechanism that keeps the germline stable.

Cellular, intracellular, and developmental expression patterns of murine SWAP-70

SWAP‐70 is part of a protein complex that catalyzes cell‐free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP‐70 in mouse tissues, sorted cells, and cultured primary cells. SWAP‐70 RNA is strongly increased upon switch‐induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP‐70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP‐70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non‐IgM isotypes. While SWAP‐70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP‐70 expression is not increased by γ or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP‐70 in immunoglobulin class switching.