OR20

Treg empowering through gene addition 

N Ayas(1) S Charbonnier(1) T Blein(1) R Thouenon(1) L Poggi(1) I André(1) S Kracker(1) J Zuber(1,2)

1:INSERM UMR 1163, Institut Imagine, Université Paris Cité, Paris, France; 2:Assistance Publique-Hôpitaux de Paris, Hôpital Necker, Service des Maladies du Rein et du Métabolisme, Transplantation rénale et Immunologie Clinique, Paris, France

Solid organ transplantation remains the most effective therapeutic option for organ failure, yet it is associated with significant complications inherent to life-long immunosuppressive therapy. CAR-Treg therapy holds promise for promoting transplant tolerance. However, CAR-Tregs are hindered by progressive attrition and dysfunction, limiting their long-term efficacy. As such, there is growing interest in stabilizing Treg identity and enhancing their suppressive function. Notably, the transcription factors BATF, JunB, and IRF4 play a critical role in driving Treg differentiation into highly suppressive cells. We hypothesized that the transgenic expression of a gain-of-function IRF4ᶠ³⁵⁹ᴸ variant, which shows enhanced cooperation with JunB, could improve CAR-Treg fitness and function. To test this, we generated IRF4ᶠ³⁵⁹ᴸ-, IRF4ʷ ͥˡ ͩ⁻ ͭʸᵖ ͤ- and empty vector-transduced Tregs from naïve Tregs via lentiviral transduction. Phenotypic and functional analyses were performed at day 16 of culture. IRF4ᶠ³⁵⁹ᴸ-expressing Tregs exhibited distinct phenotypic changes, as assessed by bulk RNA-sequencing and flow cytometry. Compared to controls, IRF4ᶠ³⁵⁹ᴸ Tregs showed increased expression of CD25, CTLA-4 and HLA-DR, indicating an activated effector phenotype. Importantly, IRF4ᶠ³⁵⁹ᴸ-expressing Tregs retained the expression of key transcription factors, including FOXP3, IKZF2, IKZF4, as well as Treg-specific epigenetic marks. In addition to this core Treg signature, IRF4ᶠ³⁵⁹ᴸ Tregs exhibited elevated production of CXCL8 under steady-state conditions, which was further enhanced in pro-inflammatory conditions. Given that myeloid-derived suppressor cells (mDSC) express CXCR1 and CXCR2, the receptors for CXCL8, we are currently investigating whether the transgenic expression of IRF4ᶠ³⁵⁹ᴸ variant in Tregs could augment their tolerogenic potential by enhancing the recruitment of mDSCs.