CD40L-targeting engineered regulatory T cells as a novel cell therapy approach for T cell mediated inflammatory diseases

Restoring immune tolerance by engineered regulatory T cell (Treg) cell therapy is a promising strategy to treat patients suffering from autoimmune and inflammatory diseases. However, in many of these conditions, relevant disease-driving antigens are unknown. Therefore, suitable target (auto-)antigens for antigen-specific Treg cell therapy are rarely available. Here, we present a novel artificial immune biosensor for Treg cells that circumvents this limitation by targeting the immune costimulatory protein CD40-ligand (CD40L), transiently expressed by activated T cells. The artificial immune receptor (AIR) comprises a CD40-derived extracellular binding domain, an intracellular costimulatory signaling domain and a T cell receptor signaling domain of the CD3-? chain. After interaction with its membrane-bound ligand, this synthetic receptor triggers a TCR-like activation program in Treg cells including induction of Treg effector molecules and cell proliferation. In a mouse model of graft-versus-host disease, transfer of Treg cells expressing the CD40-AIR significantly improved survival and demonstrated immune control of the alloantigen-reactive T cell compartment. The expression and signaling of the corresponding human CD40-AIR in human Treg cells illustrate the potential for translating this concept. Engineering Treg cells with a CD40L-sensing AIR, that detects activated T cells and, thereby, induces Treg cell activation, presents a promising therapeutic approach for a broad range of T cell mediated inflammatory diseases. Overall design: Experiment 1: anti-HLA-A2 CAR or CD40 AIR-expressing Treg cells were rested and then co-cultured with HEK cells that expressed endogenous HLA-A2 and +/- CD40L or left unstimulated. After 18 hours activated CD137 and Nr4a1 positive Treg cells were FACS-sorted and sequenced. Three biological experiments (A,B, C) were perfomed. Experiment 2: Irradiated BALBc mice that recieved bone marrow cells (2.500.000 )and spleen cells (500.000) (GvHD group), or mice that recieved250.000 Treg cells (control: truncated AIR construct or full lenght CD40 AIR construct)as treatment were sacrificed 7 days after transplantation. CD4+ T cells from spleens of these mice were FACS-sorted (10.000) and RNA-Seq was performed. n=5 mice (per group)