In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo

Novel mouse mutants with primary cellular immunodeficiencies generated by genome-wide mutagenesis.

Primary cellular immunodeficiencies are a group of genetic disorders in which 1 or more components of the cellular immune system are lacking or dysfunctional. OBJECTIVE: We sought to identify novel mouse mutants that display primary cellular immunodeficiencies. METHODS: Genome-wide N-ethyl-N-nitrosourea mutagenesis was performed in mice, followed by a phenotype screen of immunologic blood parameters. RESULTS: We identified novel mouse mutants with isolated B-cell deficiency, combined block in early B- and T-cell development, combined T-cell and natural killer cell reduction, and 3 different forms of T-cell deficiencies. One of the mutants, designated DeltaT3, displayed a combined phenotype of increased IgE, absence of peripheral T cells, and block in late thymocyte differentiation. In addition, DeltaT3 mice were unable to mount specific humoral immune responses. Chromosomal mapping and sequencing of candidate genes revealed a novel point mutation in the kinase domain of the T-cell receptor zeta chain-associated protein kinase (Zap70). In contrast to Zap70-deficient mice, DeltaT3 mutants displayed normal Zap70 mRNA and residual Zap70 protein levels. Complementation studies with Zap70-deficient mice confirmed that the point mutation found in Zap70 was causative for the DeltaT3 phenotype, including increased IgE plasma levels, a phenotype that has not been associated with altered Zap70 function in the past. CONCLUSION: Random genome-wide mutagenesis combined with a phenotype screen can be used to generate novel mouse mutants with primary cellular immunodeficiencies

Identification of immunological relevant phenotypes in ENU mutagenized mice.

The immunology screen focuses on the identification of novel gene products involved in the mammalian immune response and on the establishment of mouse models for immunological disorders. For this purpose, high throughput and semi-automated techniques were developed and optimized for low cost per sample and reproducibility. All assays are designed to be nonconsumptive and are based on peripheral blood or direct PCR amplification