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Addressing the role of PKD3 in the T cell compartment with knockout mice
Jiří Koutník1, Verena Neururer1,2, Thomas Gruber1
1Institute of Cell Genetics, Medical University Innsbruck, Innsbruck, Austria.
Background:
The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far.
Methods:
Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology.
Results:
PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells.
Conclusion:
While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.
Insights
Protein kinase D3 (PKD3) deficiency in mice enhances T cell activation and interleukin-2 production but does not affect naive T cell differentiation in vitro, suggesting a role in immune response modulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Protein kinase D3 (PKD3) is involved in T cell receptor (TCR) signaling.
- The specific role of PKD3 in primary T lymphocyte activation remains unclear.
Purpose of the Study:
- To investigate the function of PKD3 in T lymphocyte activation and immune responses.
- To elucidate the role of PKD3 in T cell differentiation and cytokine production.
Main Methods:
- RT-PCR and SDS-PAGE to assess PKD isoform expression in murine T cells.
- Analysis of PKD3-knockout mice for immune responses to immunization.
- Flow cytometry for T cell phenotyping and cytokine expression analysis.
- Luminex technology for cytokine assessment.
Main Results:
- TCR stimulation down-regulates PKD mRNA and protein levels in T cells.
- PKD3-deficient mice exhibit increased T follicular helper cell generation upon immunization.
- PKD3-deficient CD4+ T cells show enhanced interleukin-2 production ex vivo.
- No difference in in vitro differentiation of naive CD4+ T cells was observed between wild-type and PKD3-deficient cells.
- A shift towards effector/memory T cell phenotype in splenic T cells of PKD3-deficient mice at steady state.
Conclusions:
- PKD3 deficiency in vivo skews the T cell compartment towards a more activated phenotype.
- PKD3 is dispensable for naive CD4+ T cell differentiation in vitro.
- PKD3 plays a role in modulating T cell responses during immunization.
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