Background: Janus-Kinase-inhibitors (JAKi) have been approved for several indications in autoimmune diseases. Clinical studies showed that JAKi are associated with an increased risk of herpes zoster (HZ) by reactivation of varicella-zoster-virus (VZV) with a dose-dependent hazard ratio for HZ events 2 to 4. Selective JAK-1 inhibitors (JAK-1i) have been demonstrated to have a lower risk for HZ than non-selective JAKi (nsJAKi).

Objectives: So far the molecular mechanisms are unclear, how JAKi influence the VZV-specifc memory response in T-cells to set patients treated with JAKi at risk to suffer from HZ. The study aims to identify transcriptome patterns induced by administration of JAKi in vitro on lymphocytes derived from rheumatoid arthritis (RA) patients and healthy controls (HC) which are stimulated with VZV-specific antigens.

Methods: A random sample of lymphocytes derived from HC and RA patients was stimulated by VZV-specific antigens in vitro under different concentrations with selective JAK-1i filgotinib and nsJAKi tofacitinib which strongly inhibit not only JAK-1, but also JAK-2 and JAK-3. Interferon-gamma-based ELISpot assays were performed to screen for cellular reactivity against VZV-specific antigens. The immunological phenotype of VZV-specific T-cells was assessed by flow cytometry. Cytokine patterns after stimulation with VZV-specific antigens with and without JAKi were identified by multiplex cytokine analysis in cell culture supernatants. Bulk mRNA and SLAMSeq mRNA sequencing techniques were used to investigate the transcriptome pattern of VZV-specific immune cells exposed to JAKi compared to untreated controls. Western-blot-analysis was performed to analyze the phosphorylated signal-transducer-and-activator-of-transcription (STAT) proteins as an estimate of functional activity of JAK.

Results: JAKi-treated lymphocytes showed significantly lower spot-forming-unit (SFU) counts in the ELISpot assays in a concentration-dependent manner compared to untreated controls. Reduced expression of activation markers, such as cell surface marker CD69, was found in JAKi-treated cultures. Relative intensity of phosphorylated STAT1 (pSTAT1) was reduced by 81% by filgotinib and by 94% by tofacitinib using plasma-concentration-equivalent doses. In JAKi-untreated conditions, VZV-specific antigens were able to significantly induce expression of STAT1 2.4-fold, STAT2 2.8-fold, STAT3 1.2-fold and STAT4 1.7-fold. Interferon-response-factor 7 (IRF7) was induced 3.6-fold. After treatment with JAKi, STAT1 was still induced 1.5-fold after VZV-specific antigen stimulation with filgotinib, but significantly reduced by 82% in tofacitinib-treated cell cultures. JAKi-treatment significantly reduced tumor-necrosis-factor-alpha (TNFalpha) production in cell cultures stimulated with VZV-specific antigens. A similar behaviour was demonstrated by lymphocytes derived from RA patients or HC, respectively.

Conclusion: The findings demonstrated that the VZV-specific T-cell memory response after in vitro stimulation of lymphocytes was significantly diminished by treatment with JAKi. However, the impact was significantly higher in nsJAKi-treated lymphocytes than in JAK-1i-treated cell cultures. This may indicate less severe impairment regarding VZV-induced STAT-signaling and cytokine production in lymphocytes treated with selective JAK-1i compared to nsJAKi and may explain the lower HZ incidence found in JAK-1i-treated patients compared to nsJAKi-treated patients in clinical studies.

REFERENCES: NIL.

Acknowledgements: The project was funded by an unrestricted grant of the University Hospital Wuerzburg and partly supported by an investigator-initiated research granted by Galapagos.

Disclosure of Interests: Giovanni Almanzar: None declared, Felix Baum: None declared, Marie Steimer: None declared, Lydia Jahn: None declared, Sonja Hick: None declared, Alexander Ess: None declared, Christoph Rack: None declared, Igor Turin: None declared, Arne Schäfer: None declared, Panagiota Arampatzi: None declared, Thorsten Bischler: None declared, Tom Gräfenhan: None declared, Florian Erhard: None declared, Thomas Hennig: None declared, Lars Doelken: None declared, Martin Feuchtenberger: None declared, Marc Schmalzing: None declared, Martina Prelog Abbvie, Bavarian Nordic, BioNTech, Chugai-Roche, GSK, Esanum, Janssen, Novartis, Moderna, MSD, Pfizer, Sanofi, SOBI, Abbvie, BioNTech, GSK, Janssen, Novartis, Moderna, Pfizer

, Baxter, Chugai-Roche, Galapagos, GSK, MSD, Moderna, Novartis, Pfizer, SOBI