Cao et al. · Cell Reports · 2023
TRIB2 safeguards naive T cell homeostasis during aging
Cao Wenqiang, Sturmlechner Ines, Zhang Huimin, Jin Jun, Hu Bin, Jadhav Rohit R., Fang Fengqin, Weyand Cornelia M., Goronzy Jorg J.
The study
What was asked, and what was found
Cao et al., Cell Reports, 2023 asked why human naive CD8+ T cells drain away with age faster than naive CD4+ T cells do. Comparing gene expression between the two populations put TRIB2 among the top differentially expressed transcripts, more abundant in naive CD4+ cells, and the rest of the paper is the case that TRIB2 is what holds those cells in quiescence.
Every loss of function test in human cells was done with AUMsilence sdASOs from AUM BioTech, present in the medium at 2 μM with no transfection reagent, in freshly purified quiescent naive CD4+ and CD8+ T cells from blood donors. Silencing TRIB2 raised interleukin 7 driven and receptor driven proliferation, raised AKT phosphorylation at Thr308 and Ser473, and left STAT5 phosphorylation untouched, so the effect ran through AKT rather than through the interleukin 7 receptor itself. Adding an AKT inhibitor abolished the extra proliferation entirely. After 7 days with interleukin 7, around 50% of the TRIB2 silenced CD4+ cells had lost CD45RA and begun to differentiate, while most control cells stayed naive, and the silenced cells gained CD25 and produced more interleukin 2, tumour necrosis factor alpha, interferon gamma and granzyme B. Survival was unaffected.
The same reagents mapped the circuit upstream and downstream. Silencing ZBTB7B, which encodes ThPOK, lowered TRIB2, while silencing RUNX3 raised it, matching the opposite effects those two transcription factors have on the cloned TRIB2 promoter. Silencing ZBTB7B in naive CD4+ T cells reproduced the TRIB2 phenotype, and silencing RUNX3 in naive CD8+ T cells did the reverse, preserving the naive state and lowering AKT phosphorylation. Silencing both RUNX3 and TRIB2 together reversed that, placing TRIB2 downstream of RUNX3. Mouse knockout and adoptive transfer experiments, which used genetics rather than oligonucleotides, agreed. In older adults ThPOK and TRIB2 both decline in naive CD4+ T cells while RUNX3 holds steady, which the authors offer as the reason those cells lose their quiescence with age.
Key findings
- AUMsilence sdASOs at 2 μM silenced three different genes in quiescent primary human naive CD4+ and CD8+ T cells, with no transfection reagent and no effect on cell survival.(STAR Methods, Primary human cell cultures)
- Silencing TRIB2 pushed naive CD4+ T cells out of quiescence: around half lost the naive marker CD45RA after 7 days with interleukin 7, while most control cells stayed naive.(Results, TRIB2 heightens the threshold for T cell activation and effector cell differentiation)
- The mechanism was pinned down with the same reagents: silencing TRIB2 raised AKT phosphorylation without touching STAT5, and the extra proliferation it caused was abolished by an AKT inhibitor.(Results, TRIB2 inhibits AKT phosphorylation in T cells)
- Silencing the two lineage transcription factors moved TRIB2 in opposite directions, ZBTB7B down and RUNX3 up, which is how the authors placed TRIB2 downstream of both.(Results, ThPOK and RUNX3 regulate TRIB2 expression)
- Two AUMsilence sdASOs used together resolved the order of the pathway: the fall in AKT phosphorylation caused by silencing RUNX3 was undone by silencing TRIB2 in the same cells.(Results, ThPOK and RUNX3 set activation thresholds of human T cells)
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