Background/Purpose: Transposable elements (TEs) are mobile DNA elements that can replicate and move from one position to another within the host genome. Through co-evolution, TEs in the human genome have developed complex relationships with the host to regulate a variety of cellular functions. TEs are largely silent in healthy adult cells but are reactivated and transcribed in certain disease states, such as cancer, autoimmunity, and neurodegeneration. Class I TEs are retrotransposons that have mobilized throughout the genome via reverse transcription of RNA intermediates. In humans, there are two Class I TEs that encode functional endogenous reverse transcriptases (RTs), LINE-1 and HERV-K. The DNA and RNA products from TE reactivation and reverse transcription are potential source of nucleic acid that are recognized by pattern recognition receptors, such as cGAS, that sense these nucleic acids and trigger proinflammatory responses during sterile inflammation. We tested inhibitors of these endogenous RTs for their ability to block immune responses.

Methods: We generated recombinant reverse transcriptases of LINE-1 and HERV-K and developed biochemical reverse transcriptase assays. We engineered a reporter cell line to measure LINE-1 retrotranposition in cells. Utilizing these assays, we discovered potent LINE-1 RT nucleoside reverse transcriptase inhibitors (NRTIs) with a range of potencies against HERV-K RT. These inhibitors were characterized in vitro for their impact on interferon responses in immune cells and in vivo on antigen-specific T cell responses in mice.

Results: Compound A is a potent inhibitor of LINE-1 RT and a weak inhibitor of HERV-K RT. In enzymatic assays, its active tri-phosphorylated form has an IC₅₀ of 0.006 µM and 6.4 µM against LINE-1 and HERV-K RT, respectively. Compound A demonstrated excellent potency in the cell-based LINE-1 retrotransposition assay with IC₅₀ of 0.12 µM. To characterize the impact of the NRTIs on immune responses, we used Decitabine to induce LINE-1 and HERV-K expression in TREX-1 knock out THP-1 cells, and subsequently measured the effect of NRTIs on TE-induced type I interferon responses. Compound A effectively inhibited this response with IC₅₀ of 0.05 µM. We investigated the in vivo activity and mechanism of action of Compound A in a murine model of MOG peptide-induced T cell responses. Mice were immunized with MOG_35-55, 10 days later cells from draining lymph nodes were collected and cultured with MOG_35-55. At 40 mg/kg oral dosing, Compound A suppressed antigen specific T cell proliferation and reduced IL-17A, TNFa and IFNg production.

Conclusion: We used biochemical and cell-based assays to identify potent endogenous RT inhibitors. These inhibitors suppressed in vitro repeat element-induced type I interferon responses in monocytic cells and attenuated in vivo antigen-specific T cell responses. These data demonstrate the potential of endogenous RT inhibitors to modulate host immune responses for therapeutic benefit in autoimmune/inflammatory diseases.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibitors-of-endogenous-reverse-transcriptases-suppress-in-vitro-type-i-interferon-responses-and-in-vivo-antigen-specific-t-cell-responses/