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J Roes

Publications and source records attributed to J Roes.

14 recordsLinked to original sources
PubMed1998

Antiviral protection and germinal center formation, but impaired B cell memory in the absence of CD19.

Coligation of CD19, a molecule expressed during all stages of B cell development except plasmacytes, lowers the threshold for B cell activation with anti-IgM by a factor of 100. The cytoplasmic tail of CD19 contains nine tyrosine residues as possible phosphorylation sites and is postulated to function as the signal transducing element for complement receptor (CR)2. Generation and analysis of CD19 gene-targeted mice revealed that T cell-dependent (TD) antibody responses to proteinaceous antigens were impaired, whereas those to T cell-independent (TI) type 2 antigens were normal or even augmented. These results are compatible with earlier complement depletion studies and the postulated function of CD19. To analyze the role of CD19 in antiviral antibody responses, we immunized CD19(-/-) mice with viral antigens of TI-1, TI-2, and TD type. The effect of CD19 on TI responses was more dependent on antigen dose and replicative capacity than on antigen type. CR blocking experiments confirmed the role of CD19 as B cell signal transducer for complement. In contrast to immunization with protein antigens, infection of CD19(-/-) mice with replicating virus led to generation of specific germinal centers, which persisted for >100 d, whereas maintenance of memory antibody titers as well as circulating memory B cells was fully dependent on CD19. Thus, our study confirms a costimulatory role of CD19 on B cells under limiting antigen conditions and indicates an important role for B cell memory.

Animals↗
PubMed1997

B lymphocyte-specific, Cre-mediated mutagenesis in mice.

Adaptation of the P1 phage-derived Cre /loxP site- specific recombination system to the gene targeting technique allows for the conditional deletion of genes in mice. To selectively modify genes in B lymphocytes, we have generated mice (designated CD19-Cre) which express cre under the transcriptional control of the B lineage-restricted CD19 gene. In a model system involving the cross of CD19-Cre mice with mice bearing a loxP -flanked substrate, we find a deletion efficiency of 75-80% in bone marrow-derived pre-B cells that increases to 90-95% in splenic B cells.

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PubMed1997

Targeted disruption of the V(H) 81X gene: influence on the B cell repertoire.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

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PubMed1995

Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice.

CD19 is the hallmark differentiation antigen of the B lineage. Its early expression has implicated a role for CD19 during the antigen-independent phases of B-cell development, whereas in mature B cells CD19 can act synergistically with surface immunoglobulin to induce activation. We have generated CD19-deficient mice and found that development of conventional B cells is unperturbed. However, mature CD19-/- B cells show a profound deficiency in responding to protein antigens that require T-cell help. This is accompanied by a lack of germinal centre formation and affinity maturation of serum antibodies. Thus CD19 is crucial for both initial B-cell activation by T-cell-dependent antigens and the maturation and/or selection of the activated cells into the memory compartment. An impairment in ligand-driven selection may also be responsible for the observation of a striking reduction in the B-1 (formerly Ly-1) B-cell subset, thought to develop under the control of self-antigens and bacterial antigens (reviewed in ref. 2).

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PubMed1995

Mouse anti-mouse IgD monoclonal antibodies generated in IgD-deficient mice.

The production of high-affinity monoclonal antibodies (mABs) is generally restricted to antigens recognised as foreign by the immune system. Here we report the generation of mouse mABs specific for mouse IgD. Mice rendered IgD-deficient by gene targeting and consequently immunologically fully responsive to mouse IgD, were used to elicit a humoral response against mouse IgD. Hybridomas producing mABs of high affinity were isolated and clones specific for non-allotypic determinants on the Fc or Fab portion of mouse IgD were obtained. The data show that mice lacking a protein of interest due to targeted gene inactivation can be utilised for the production of high-affinity mABs specific for that mouse protein and should also facilitate the generation of mABs specific for proteins highly conserved between species.

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PubMed1994

Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning.

Neural-cell adhesion molecules (N-CAMs) are members of the immunoglobulin superfamily mediating homo- and heterophilic cell-cell interactions. N-CAM exists in various isoforms which are generated by alternative splicing. During embryonic development, N-CAMs are expressed in derivatives of all three germ layers, whereas in the adult animal they are predominantly present in neural tissue. Processes like neurulation, axonal outgrowth, histogenesis of the retina and development of the olfactory system are correlated with the regulated expression of N-CAMs. We show here that N-CAM-deficient mice generated by gene targeting appear healthy and fertile, but adult mutants show a 10% reduction in overall brain weight and a 36% decline in size of the olfactory bulb. N-CAM deficiency coincides with almost total loss of protein-bound alpha-(2,8)-linked polysialic acid, a carbohydrate structure thought to be correlated with neural development and plasticity. The animals showed deficits in spatial learning when tested in the Morris water maze, whereas activity and motor abilities appeared normal.

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PubMed1993

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells.

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD-deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell-independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen-driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens.

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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.

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PubMed1991

Cell autonomous expression of IgD is not essential for the maturation of conventional B cells.

To analyse the function of IgD in vivo, we generated a 'loss of function' mouse model utilizing gene targeting technology. By homologous recombination in a (C57BL/6 x CBA)F1 mouse embryonic stem cell (ES) line one allele of the delta heavy chain gene was rendered non-functional. In chimeric mice obtained after injection of the targeted ES cells into blastocysts derived from severe combined immunodeficient mice we analysed ES cell derived B lymphocytes expressing the targeted or the wild-type allele by using allotype specific reagents. We show that B cells expressing the targeted allele appear in the periphery as IgM+ D- cells at normal frequency. They express the CD23 marker and respond to a T cell dependent antigen. Thus, cell autonomous expression of IgD is neither essential for B cell maturation into an antigen responsive state nor for antigen dependent triggering of the cells into an immune response.

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PubMed1989

V gene rearrangement is required to fully activate the hypermutation mechanism in B cells.

Nonproductively rearranged H and L chain loci of B cell hybridoma lines expressing heavily mutated antibodies were cloned and partially sequenced. The results confirm earlier data showing that somatic point mutations are as frequent in nonproductively rearranged loci containing a rearranged V gene as in productively rearranged loci. They establish in addition that in nonproductive H chain loci which bear a DJH rearrangement the frequency of somatic mutations is more than 10 times lower (0.2%) than in VDJH loci expressed by the same cells (2.5%). Thus, the hypermutation mechanism operating in B cell differentiation is targeted at V genes rearranged to the J locus and may require nucleotide sequences associated with both V and J elements in order to be fully activated. An inversion of the JH2 segment was detected in one DJH locus. This inversion appears to be the result of a secondary joining event occurring occasionally in the course of B cell development.

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PubMed1987

Timing, genetic requirements and functional consequences of somatic hypermutation during B-cell development.

While somatic antibody mutants are rare in the preimmune repertoire and in primary immune responses, they dominate secondary and hyperimmune responses. We present evidence that somatic hypermutation is restricted to a particular pathway of B-cell differentiation in which distinct sets of B-cell clones are driven into the memory compartment. In accord with earlier results of McKean et al. (1984) and Rudikoff et al. (1984), somatic mutation occurs stepwise in the course of clonal expansion, before and after isotype switch, presumably at a rate close to 1 X 10(-3) per base pair per generation. At this rate, both selectable and unselectable mutations accumulate in the rearranged V region genes. The distribution of replacement mutations in the V regions shows that a fraction of the mutations in CDRs is positively selected whereas replacement mutations are counterselected in the FRs. By constructing an antibody mutant through site-specific mutagenesis we show that a point mutation in CDR1 of the heavy chain, found in most secondary anti-NP antibodies, is sufficient to increase NP binding affinity to the level typical for the secondary response. Somatic mutation may contribute to the immune repertoire in a more general sense than merely the diversification of a specific response. We have evidence that clones producing antibodies which no longer bind the immunizing antigen can be kept in the system and remain available for stimulation by a different antigen. Somatic mutations are 10 times less frequent in DJH loci than in either expressed or non-expressed rearranged VDJH or VJ loci. We therefore conclude that a V gene has to be brought into the proximity of the DJH segment in order to fully activate the hypermutational mechanism in these loci.

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