Novel mutant mouse line emphasizes the importance of protein kinase C theta for CD4⁺ T lymphocyte activation.

ABSTRACT:

Background

The protein kinase C theta (PKC?) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKC? is not only essential for activation of NF-?B, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKC? was analyzed for its ability to stimulate T lymphocyte activation.

Methods

T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKC? expression constructs. A mouse line expressing mutated instead of wild type PKC? was analyzed in comparison to PKC?-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry.

Results

In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKC?. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKC?-E2^mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKC? combined with reduced PKC? protein levels in CD4⁺ T lymphocytes. Thus, PKC?-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4⁺ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKC?-E2^mut mice show a phenotype similar to conventional PKC?-deficient mice.

Conclusion

Taken together, PKC?-E2^mut mice show a phenotype similar to conventional PKC?-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKC?-E2-mutant mouse line independently validate the importance of PKC? downstream of the antigen-receptor complex for activation of CD4⁺ T lymphocytes.

SUBMITTER: Siegmund K 

PROVIDER: S-EPMC6537413 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature