Treg Empowering Through Gene Addition
AYAS N. 1, CHARBONNIER S. 1, BLEIN T. 1, THOUENON R. 1, POGGI L. 1, ANDR╔ I. 1, KRACKER S. 1, ZUBER J. 1,2
1 Institut Imagine, Paris, France; 2 H˘pital Necker, Paris, France
Solid organ transplantation remains the best therapeutic option for life-threatening organ failure, yet is associated with severe complications inherent to life-long immunosuppressive regimen. CAR-Treg therapy appears to be a promising strategy to promote tolerance towards allograft. However, similarly as CAR-T cells, CAR-Tregs face the issue of progressive attrition and dysfunction, limiting their long-term efficacy. Hence, the community is embracing the challenge of further promoting the stability of Treg cell identity, and of enhancing their suppressive capacities. Notably, BATF, JunB, and IRF4 transcription factors play a key role for Tregs differentiation especially towards highly suppressive cells commitment, while BATF or c-Jun overexpression skew CAR T cells away from exhaustion. We hypothesized that transgenic expression of an IRF4F359L gain-of-function variant, which demonstrated an increased activity on Jun-dependent binding site, could enhance CAR-Treg fitness and function.
IRF4F359L-, IRF4wild-type- and empty vector- Tregs were generated from naïve Tregs through lentiviral transduction. Phenotypic characterization and functional testing were performed at day 16 of culture.
IRF4F359L-expressing Tregs demonstrated an increased expression of CD25 that correlated with enhanced phosphorylation of STAT5. Notably, improved IL-2 signaling imparted IRF4F359L-Tregs with enhanced IL-2-dependent fitness, when assessed in a competitive co-cultivation assay with control Tregs. More strikingly, IRF4F359L-Tregs acquired hallmark markers of effector Tregs, including high expression of HLA-DR. In line with this finding, IRF4F359L-Tregs elicited greater in vitro suppression than IRF4WT-Tregs.
Together, our preliminary data identifies transgenic expression of IRF4F359L variant as a promising strategy to empower human Tregs in a therapeutic purpose. In vivo suppressive capacities will be soon evaluated in a xenogeneic graft-versus-host disease (GVHD) model, induced by the transfer of human peripheral blood lymphocytes (PBL) into NSG mice. We will evaluate the impact of the variant in Tregs and CAR-Tregs, that delay or prevent the course of GVHD, respectively, when co-transferred with PBL.