PRELI is a mitochondrial regulator of human primary T helper cell apoptosis, STAT6 and Th2 cell differentiation

Tahvanainen J, Kallonen T, Lahteenmaki H, Heiskanen KM, Westermarck J, Rao KV, Lahesmaa R
Source: Blood
Publication Date: (2008)
Issue: epub: online
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
Buffy coat CD4+ cells were nucleofected with pIRES-H2Kk-PRELI or pIRES-H2Kk vectors. Transfected cells were incubated at 37°C for 16h (2.5x106 cells/ml) after which dead cells were removed and the transfected cells were enriched by MACS. Briefly, dead cells, apoptotic cells and debris were depleted from the cultures by using MACS Dead Cell Removal Microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Subsequently, the remaining cells were incubated with MACSelect Kk MicroBeads coated with H-2Kk-antibody (Miltenyi Biotech) for 15min to magnetically label the transfected cells. The magnetic separation of H-2Kk-positive cells was done with a positive selection column placed in the magnetic field of a MACS separator. After the enrichment, cell viability and purity (H-2Kk-positivity) were determined by flow cytometry. The purified H-2Kk-positive cells were activated. In PRELI knockdown studies, naive or peripheral blood CD4+ cells, nucleofected with scrambled or PRELI-specific siRNA oligos (1.5μg / 4x106 cells / transfection) were incubated at 37°C for 24h (2x106 cells/ml) and subsequently activated.
The identification of novel factors regulating human T helper (Th) cell differentiation into functionally distinct Th1 and Th2 subsets is important for understanding the mechanisms behind human autoimmune and allergic diseases. We have identified PRELI, a novel protein that induces oxidative stress and mitochondrial apoptosis pathway in human primary Th cells. We also demonstrate that PRELI inhibits Th2 cell development and downregulates STAT6, a key transcription factor driving Th2 differentiation. Our data suggest that Calpain, an oxidative stress induced cysteine protease, is involved in the PRELI-induced downregulation of STAT6. Moreover, we observed that a strong T cell receptor (TCR) stimulus induces expression of PRELI and inhibits Th2 development. Our results suggest that PRELI is involved in a mechanism wherein the strength of the TCR stimulus influences the polarization of Th cells. This study identifies PRELI as a novel factor influencing the human primary Th cell death and differentiation.