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Biomedical subjects

K Rajewsky

Publications and source records attributed to K Rajewsky.

At least 19 recordsLinked to original sources
PubMed1992

Natural occurrence and origin of somatically mutated memory B cells in mice.

While most murine peripheral B cells express germline-encoded antibodies of classes M and D (mu+ delta+ cells), small numbers of memory B cells expressing somatically mutated immunoglobulin G antibodies are generated upon T cell-dependent immunization. Analyzing the antibody repertoire of the mu-delta- B cell pool in unimmunized mice, we show that these cells express somatically mutated VH genes and that most of these genes derive from a set of germline VH genes dominantly expressed by mu+delta+ B cells. Thus, class-switched memory B cells are generated in the absence of intentional immunization, presumably in response to environmental antigens. These cells are either recruited from mu+delta+ B cells or selected from newly arising B cells in parallel to the latter, by the same antigens.

Amino Acid Sequence↗
PubMed1992

T helper cell membranes promote IL-4-independent expression of germ-line C gamma 1 transcripts in B cells.

Studies using plasma membranes from activated Th cell clones (Th membranes) to stimulate B cells have shown that both a contact-mediated activation signal plus Th-derived cytokines are required for antibody production. In order to clearly separate and define the role of these two signals in isotype switching, B cells were stimulated with Th membranes in the presence or absence of cytokines, and the transcriptional activity of the unrearranged H chain loci was determined. In the presence of Th membranes, two known switch factors were shown to specifically induce germ-line transcription of the same H chain loci as in LPS-stimulated B cells (IL-4 induced C gamma 1 and C epsilon transcription, transforming growth factor-beta induced C alpha transcription). The contact-mediated activation signal provided by the Th membranes, in the absence of any added cytokines, resulted in the specific induction of C gamma 1 germ-line transcription, and thus functioned as a switch signal for IgG1. These findings provide a mechanism for previously observed IL-4-independent isotype switching to IgG1.

Animals↗
PubMed1992

A critical role of lambda 5 protein in B cell development.

The lambda 5 gene is a homolog of immunoglobulin J lambda-C lambda genes, expressed specifically in immature B-lineage cells. Lambda 5-encoded molecules form membrane complexes with mu or D mu proteins in association with an additional protein specifically expressed in immature B cells that is encoded by the Vpre-B gene. We have generated mice in which the lambda 5 gene is inactivated by targeted gene disruption in embryonic stem cells. In these mice, B cell development in the bone marrow is blocked at the pre-B cell stage. However, the blockade is leaky, allowing B cells to populate the peripheral immune system at a low rate. These cells are allelically excluded and able to respond to antigen.

Animals↗
PubMed1992

T cell-dependent antibody production by Ly-1 B cells.

Through the use of a SCID transfer system, we have demonstrated that under certain conditions, the production of Ig by Ly-1 B cells can be modulated by T cells. This modulation can take the form of enhanced isotype production or isotype-switch induction and to some extent appears to be dependent on the activation state of the T cells. Furthermore we have shown that Ly-1 B cells can mount an idiotypically restricted T cell-dependent immune response to the antigen PC-KLH. This result suggests that the previous failure to observe T cell-dependent responses by Ly-1 B cells has been due to these B cells being "blind" to the antigens used and is not due to some inherent property of these B cells. When one considers the previous reports of the substantial contribution of Ly-1 B cells to the natural serum immunoglobulin levels and the ability of T cells to affect Ig production by Ly-1 B cells documented in this report, it is clear that the interaction of T cells with the Ly-1 B-cell population is important in determining the "natural" serum Ig repertoire of the mouse.

Animals↗
PubMed1992

Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion.

Burnet's clonal selection theory suggests that each B lymphocyte is committed to a single antibody specificity. This is achieved by a programme of somatic rearrangements of the gene segments encoding antibody variable (V) regions, in the course of B-cell development. Evidence from immunoglobulin-transgenic mice and immunoglobulin-gene-transfected transformed pre-B cells suggest that the membrane form of the immunoglobulin heavy (H) chain of class mu (microns), expressed from a rearranged H-chain (IgH) locus, may signal allelic exclusion of the homologous IgH locus in the cell and initiation of light (L)-chain gene rearrangement in the Ig kappa loci. We report here that targeted disruption of the membrane exon of the mu chain indeed results in the loss of H-chain allelic exclusion. But, some kappa chain gene rearrangement is still observed in the absence of micron expression.

Alleles↗
PubMed1992

Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen.

The accumulation of somatic mutants in splenic B lymphocytes early after primary immunization with the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) coupled to chicken gamma globulin (CG) was determined. Rearranged V186.2 heavy chain genes were amplified by the polymerase chain reaction from genomic DNA and subjected to nucleotide sequence analysis. Somatic antibody mutants become detectable on day 6 after immunization, and most of the somatic mutations accumulating in the memory compartment are introduced until day 14. At this time strong selection for mutants expressing high binding affinity for NP is apparent. Extrapolation from the mutation frequency increases between day 6 and day 14 to the previously determined mutation frequency at week 6 (Weiss. U. and Rajewsky, K., J. Exp. Med. 1990, 172: 1681) leads to the prediction that the process of mutant generation ceases to operate around day 22 after primary immunization.

Amino Acid Sequence↗
PubMed1992

An explanation for the defect in secretion of IgM Mott cells and their predominant occurrence in the Ly-1 B cell compartment.

Mott cells are a variant form of plasma cell in which the immunoglobulin (Ig), rather than being secreted, accumulates in rough endoplasmic reticulum-derived vesicles called Russell bodies. We have examined the molecular cause of this defect and the in vivo origin of IgM Mott cells. Our examination of the Ig variable region gene sequences of two IgM Mott hybridomas derived from C.B-20 Ly-1 B cells showed all to be germ line. In a series of mix and match transfection experiments, the Mott phenotype was only reconstituted when the original Mott specificity was expressed as an IgM, suggesting that both the specificity and the isotype were critical to the formation of Russell bodies. Based on our finding that Russell body formation was dependent on the Ig isotype being IgM, we suggest that the Mott phenotype is apparent only after differentiation of B cells into plasma cells and that probably the major cause of the IgM Mott phenotype is low-affinity interaction of the Mott Ig with some as yet unknown intracellular component(s) being stabilized by the intrinsic high avidity of the pentameric secreted form of IgM. Consistent with this proposal was the finding that after in vitro lipopolysaccharide (LPS) stimulation of sorted Ly-1 B cells derived from C.B-20 mice, Mott cells represented up to 5% of the IgM plasma cells in the culture. LPS stimulation of conventional B cells also induced the appearance of IgM Mott cells, but at the much reduced level of 0.1%, suggesting that the major, if not the only, source of Mott cells in vivo is the Ly-1 B cell population. A possible causal relationship between the elevated frequency of Mott cells in the Ly-1 B cell-derived LPS blasts and the repertoire selection inherent in the development of these B cells is discussed.

Amino Acid Sequence↗
PubMed1992

Immunoglobulin heavy and light chain gene sequences of a human CD5 positive immunocytoma and sequences of four novel VHIII germline genes.

To analyse the V genes expressed by an IgM lambda CD5-positive immunocytoma heavy and light chain V region genes were cloned and sequenced. The heavy chain is composed of a previously undescribed VHIII gene joined to an unknown D gene and to JH4. The light chain V region is composed of a V lambda II gene rearranged to J lambda 1. In an attempt to clone the germline counterpart of the VHIII gene expressed in the immunocytoma PCR amplifications of genomic DNA were carried out and four previously unknown VHIII genes were identified. As several independent clones for the heavy and light chain V region genes were sequenced the rate of somatic mutation of the V genes was calculated to be below 2 x 10(-5)/bp/cell division.

Amino Acid Sequence↗
PubMed1992

Early and late B-cell development in the mouse.

A common principle in B-cell development is the stringent selection of cells expressing appropriate antibody V regions as surface receptors. Cells failing to do so appear destined to rapid death. These life-death decisions are mediated by signals whose nature is not yet understood but whose generation involves immunoglobulin receptor complexes on B cells and B-cell progenitors.

Animals↗
PubMed1992

A functional antibody mutant with an insertion in the framework region 3 loop of the VH domain: implications for antibody engineering.

We have studied the effects of a four residue insertion into the FR3 loop of the heavy chain variable region from the anti-NP antibody B1-8. The insertion mutant is obtained as secreted antibody without major defects in biosynthesis, indicating that antibody variable domains can accommodate length variation not only in complementarity determining regions (CDRs), but also in framework region (FR) loops. The B1-8 antigen binding site is not affected by the change in a neighbouring loop. FR3 insertions represent a new method of antibody engineering with a potential to obtain strong antigen binding by designing additional antigen contacting residues.

Amino Acid Sequence↗
PubMed1992

[Detection of clonal immunoglobulin rearrangements in paraffin embedded tissues by PCR].

A polymerase chain reaction based method was established using immunoglobulin VH and VK gene rearrangements as markers to detect clonal B cell populations in paraffin embedded sections. Family specific VH and VK primers are used in separate reactions together with the corresponding J primers to amplify rearranged VH and VK genes from genomic DNA. This allows to distinguish clonal from polyclonal B cell populations in most of the cases. The method may be helpful in routine diagnosis of B cell NHL and some morphological and immunohistochemical difficult cases.

Base Sequence↗
PubMed1991

Intraclonal generation of antibody mutants in germinal centres.

The generation and selection of somatic antibody mutants are key elements of acquired immunity, essential for the affinity maturation of antibody responses dependent on T cells. The mutants are generated through a mechanism that introduces point mutations at high rate into rearranged variable (V) region genes in the course of cell proliferation. Their appearance coincides with the generation of germinal centres, which are characterized by oligoclonal B-cell proliferation and have been suggested to be the microenvironment in which antibody mutants are generated. We report here direct evidence for this hypothesis. Rearranged V-region genes were amplified from the genomic DNA of cells picked from individual germinal centres. The sequence analysis of these genes revealed that most represent cells of distinct B-cell clones which expanded locally, generating somatic antibody mutants at high rate. By contrast, antigen-induced proliferation of B cells at another site, periarteriolar lymphocyte sheath-associated foci, was not associated with somatic hypermutation.

Amino Acid Sequence↗
PubMed1991

Generation and analysis of interleukin-4 deficient mice.

Interleukin-4 (IL-4) promotes the growth and differentiation of many hematopoietic cells in vitro; in particular, it directs the immunoglobulin (Ig) class switch to IgG1 and IgE. Mice homozygous for a mutation that inactivates the IL-4 gene were generated to test the requirement for IL-4 in vivo. In the mutant mice T and B cell development was normal, but the serum levels of IgG1 and IgE were strongly reduced. The IgG1 dominance in a T cell-dependent immune response was lost, and IgE was not detectable upon nematode infection. Thus, some but not all of the in vitro properties of IL-4 are critical for the physiology of the immune system in vivo.

Aging↗
PubMed1991

Most peripheral B cells in mice are ligand selected.

Using amplified cDNA and genomic libraries, we have analyzed the VH gene repertoire of pre-B cells and various B cell subsets of conventional mice at the level of VH genes belonging to the J558 VH gene family. The sequence data were evaluated on the basis of a newly established list of 67 J558 VH genes that comprise approximately two-thirds of the J558 VH genes of the murine IgHb haplotype. The results of the analysis demonstrate that VH gene utilization in pre-B cells, although biased to some extent by B cell autonomous VH gene selection, scatters over the whole range of J558 VH genes present in the germline. In contrast, in mature, peripheral B cells comprising long-lived mu + delta high B cells as well as Ly-1 B cells, small overlapping sets of germline VH genes are dominantly expressed. The data indicate that the recruitment of newly generated B cells into the long-lived peripheral B cell pool is mediated through positive selection by internal and/or external antigens. Because of the absence of immunoglobulin class switching and somatic hypermutation, this process is different from the selection of memory B cells in T cell-dependent immune responses.

Animals↗
PubMed1991

B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames.

In productively rearranged murine VH-DH-JH genes (encoding immunoglobulin heavy chain variable regions), the DH elements are preferentially used in one particular reading frame (RF1), although the recombination breakpoints at the DH-JH border vary. Despite this variability, the bias of RF usage is not due to cellular selection by antigen but is quantitatively established at the stage of DH-JH rearrangement: RF3 is counterselected on the basis of stop codons. RF2 allows the expression of a truncated mu chain (D mu protein) from most DH-JH joints. Using B cells in which the membrane exon of the mu chain is disrupted by homologous recombination on one of the two homologous chromosomes, we obtain evidence that membrane-bound D mu signals arrest of differentiation, presumably by preventing VH-DHJH joining. In addition to RF3 and RF2 counterselection, promotion of DH-JH joining in areas of sequence homology further enforces RF1 usage.

Amino Acid Sequence↗
PubMed1991

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene.

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.

Animals↗