Burattin et al. · Science Advances · 2024

LINE1 modulate human T cell function by regulating protein synthesis during the life span

Burattin Filippo V., Vadalà Rebecca, Panepuccia Michele, Ranzani Valeria, Crosti Mariacristina, Colombo Federico A., Ruberti Cristina, Erba Elisa, Prati Daniele, Nittoli Teresa, Montini Giovanni, Ronchi Andrea, Pugni Lorenza, Mosca Fabio, Ricciardi Sara, Abrignani Sergio, Pietrasanta Carlo, Marasca Federica, Bodega Beatrice

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The study

What was asked, and what was found

Burattin et al., Science Advances, 2024 asked why a newborn's T cells react faster than an adult's, and followed the answer to a repeat family that most work treats as genomic noise. The group had already shown that long interspersed nuclear element 1 sequences are spliced into non-canonical transcript variants that hold adult naive CD4+ T cells in quiescence. Here they found that neonatal naive CD4+ T cells carry almost none of those transcripts, because tonic T cell receptor signalling from self-antigen keeps mTORC1 active and PTBP1 suppresses the splicing that produces them.

The causal tests were done with self-delivering AUM antisense oligonucleotides, given to quiescent primary human CD4+ T cells at 10 μM with no transfection reagent, for 48 hours and then maintained at the same concentration through activation. Three RNAs were silenced with separate oligonucleotides, against an unrelated scramble control. Silencing PTBP1 with an AUMsilence sdASO brought LINE1 and the LINE1 transcript variants back in neonatal cells and left other repeat families and other PTBP1 splice targets untouched (P = 0.006 by paired two-tailed t test, and P < 0.001 with F = 20.8 by two-way ANOVA, n = 3). Silencing LINE1 itself in adult CD4+ T cells raised the rate of protein synthesis (P = 0.04, n = 4) and pushed more cells into S, G2 and M (P = 0.009, F = 11.2, n = 3). Silencing a third target, HERVK, changed neither readout, which is the specificity control that makes the LINE1 result stand.

The same reagents then carried the story to the other end of life. LINE1 expression rose steadily from newborns through toddlers, children and adults, and peaked in donors over 80, where protein synthesis in both naive and memory CD4+ T cells was lower than in adults. Silencing LINE1 in those elderly T cells raised protein synthesis again (P = 0.03). The authors put LINE1 expression forward as a quantitative reading of how well a human T cell can function at any age. Every one of these experiments was done in quiescent primary human T cells, and none of them used a transfection reagent.

Key findings

Self-delivering AUM antisense oligonucleotides silenced three separate RNAs in quiescent primary human CD4+ T cells at 10 μM with no transfection reagent.(Methods, T cell treatments)
Silencing PTBP1 with an AUMsilence sdASO in neonatal naive CD4+ T cells brought LINE1 and the LINE1 transcript variants back, and left other repeat families and other PTBP1 splice targets untouched, which is what established PTBP1 as the splicing suppressor in this circuit.(Results, In neonatal naive CD4+ T cells, LINE1 expression is suppressed by mTORC1 activity via PTBP1, Fig. 3, G to I)
Silencing LINE1 in adult CD4+ T cells raised the rate of protein synthesis and pushed more cells into S, G2 and M, which is the causal step tying LINE1 expression to how fast a T cell can respond.(Results, LINE1 expression regulates the rate of protein synthesis and cell cycle progression in CD4+ T cells)
Silencing a second target, HERVK, changed neither protein synthesis nor cell cycle progression, so the LINE1 result was specific rather than a general effect of the oligonucleotide.(Results, LINE1 expression regulates the rate of protein synthesis and cell cycle progression in CD4+ T cells)
In naive and memory CD4+ T cells from donors over 80, where LINE1 has accumulated and protein synthesis has fallen, silencing LINE1 raised protein synthesis again.(Results, LINE1 are overexpressed in T cells during aging, leading to a reduced rate of protein synthesis)

Product: AUMsilence sdASO
Targets: PTBP1 · human · mRNALINE1 · human · non-coding RNA, spliced transcript variants of long interspersed nuclear element 1HERVK · human · non-coding RNA, human endogenous retrovirus K transcript
Models: human naive CD4+ T cells sorted from umbilical cord blood of full-term neonates · primary cellshuman naive and memory CD4+ T cells from adult and elderly donors, across an age series from newborns to individuals over 80 · primary cells
Delivery: added to the culture medium at 10 μM with no transfection reagent, for 48 hours, and kept at 10 μM through the subsequent activation
Readouts: RT-qPCR, RNA-seq with gene set enrichment analysis, puromycin incorporation for protein synthesis, flow cytometry cell cycle analysis, immunoblot, RNA immunoprecipitation, RNA fluorescence in situ hybridisation
Measured: oligonucleotide concentration and exposure used throughout, in quiescent primary human T cells: 10 μM for 48 hours, then maintained at 10 μM during activation (no transfection reagent, complete RPMI with recombinant interleukin 2 at 200 IU/ml)re-expression of LINE1 after silencing PTBP1 in neonatal naive CD4+ T cells: P = 0.006 for LINE1 in panel H, where HERVK, MaLR, AluY and alpha-Satellite carry no significance mark; P < 0.001 with F = 20.8 for the LINE1-transcripts in panel I (n = 3 individuals)protein synthesis in adult CD4+ T cells after silencing LINE1, by puromycin incorporation: P = 0.04 (n = 4 individuals, cells activated for 24 hours)cell cycle progression in adult CD4+ T cells after silencing LINE1: P = 0.009, F = 11.2 (n = 3 individuals, cells activated for 72 hours)protein synthesis in naive and memory CD4+ T cells from elderly donors after silencing LINE1: P = 0.03 (naive n = 3, memory n = 1, cells activated for 24 hours)knockdown of LINE1 after it had been allowed to re-express in neonatal naive T cells: P = 0.05 for scramble control against LINE1 oligonucleotide (n = 4 individuals)
Figures: Fig. 3, G to I Silencing PTBP1 in neonatal naive CD4+ T cells and the resulting return of LINE1 and of the LINE1 transcript variants, with other repeat families unchanged.Fig. 5 The functional consequence of silencing LINE1: protein synthesis by puromycin incorporation and cell cycle progression, in adult T cells and in neonatal T cells where LINE1 had first been allowed to return.Fig. 6 LINE1 expression across the human life span from newborns to donors over 80, and the recovery of protein synthesis when LINE1 is silenced in elderly T cells.
Citation: Burattin et al. LINE1 modulate human T cell function by regulating protein synthesis during the life span. Science Advances 2024;10:eado2134. doi:10.1126/sciadv.ado2134

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