Marasca et al. · Nature Genetics · 2022
LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion
Marasca Federica, Sinha Shruti, Vadalà Rebecca, Polimeni Benedetto, Ranzani Valeria, Paraboschi Elvezia Maria, Burattin Filippo Vittorio, Ghilotti Marco, Crosti Mariacristina, Negri Maria Luce, Campagnoli Susanna, Notarbartolo Samuele, Sartore-Bianchi Andrea, Siena Salvatore, Prati Daniele, Montini Giovanni, Viale Giuseppe, Torre Olga, Harari Sergio, Grifantini Renata, Soldà Giulia, Biffo Stefano, Abrignani Sergio, Bodega Beatrice
The study
What was asked, and what was found
Marasca et al., Nature Genetics, 2022 asked what holds a naive human T cell in its resting state, and found the answer in a class of RNA nobody was reading as regulatory. Long interspersed nuclear element 1 sequences sitting inside introns are spliced out as exons into non-canonical variants of ordinary protein-coding genes. The authors identified 461 such transcripts from 407 genes. In naive CD4+ T cells these variants are made under the control of the transcription factor IRF4, held at chromatin by nucleolin, and there they suppress H3K36me3 and keep the corresponding genes stalled. Activating the cell dismantles the arrangement, through the splicing suppressor PTBP1 and the elongation factor GTF2F1.
Every step of that mechanism was tested by knockdown, and the knockdowns are what make this study relevant to AUM. Seven targets were silenced with AUMsilence sdASOs: the LINE1-containing transcripts themselves, the two specific variants HIRA.L1 and RAB22A.L1, and the messenger RNAs for IRF4, nucleolin, GTF2F1 and PTBP1. Between two and five oligonucleotides were pooled per target, mixed in equimolar proportions. The methods state the delivery in plain words: given without any transfection reagent, by gymnosis, at 10 μM. The cells were primary human T cells taken from healthy donors.
The result the paper is built around comes from patients. Memory CD4+ and CD8+ tumour-infiltrating lymphocytes isolated from colorectal cancer and non-small cell lung cancer samples had re-expressed LINE1 in every patient examined. Knocking those transcripts down in the patient cells lowered the fraction carrying PD-1, LAG-3 and TIM3, raised interferon gamma and granzyme B, and boosted their ability to kill heterologous monocytes, which is what the authors mean when they write that depleting the transcripts restores TIL effector function. The proliferation capacity of exhausted T cells was not restored, which the authors report alongside the rest.
Key findings
- The oligonucleotides were given by gymnosis into primary human T cells, with no transfection reagent of any kind.(Methods, Knockdown experiments)
- Knocking down the LINE1-containing transcripts in tumour-infiltrating lymphocytes taken from patients reduced the exhaustion receptors on their surface and raised their effector cytokines.(Results, LINE1-containing transcripts suppress the effector function of TILs)
- Those cells also killed better after the knockdown, so the change reached function and not only surface markers.(Results, LINE1-containing transcripts suppress the effector function of TILs)
- The paper's headline claim is that depleting these transcripts restores the effector function of a patient's tumour-infiltrating lymphocytes, and the oligonucleotide is what demonstrates it.(Abstract)
- The authors read that restoration as evidence that the accumulation is not a fixed state but a reversible mechanism.(Discussion)
- The same approach mapped the mechanism upstream, since knocking down the transcription factor IRF4 and the chromatin anchor nucleolin separated the two steps that keep these transcripts in place.(Results, end of the exhausted T cell section)
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