Conferring alloantigen specificity to regulatory T cells: a comparative analysis of cell preparations undergoing clinical development in transplantation.

Conferring alloantigen-specificity to ex vivo expanded CD4+CD25+Foxp3+ Tregs increases their capacity to counteract effector alloimmune responses following adoptive transfer into transplant recipients. Three strategies are currently undergoing clinical development, which involve: 1) expanding Tregs in the presence of donor B cells (donor alloantigen-reactive (DAR-Tregs)); 2) culturing Tregs with donor cells in the presence of co-stimulation blockade (CSB-Tregs); and 3) transducing Tregs with an HLA-A2-specific chimeric antigen receptor (CAR-Tregs). Our goal here was to assess the relative potency of each of these manufactured Treg products both in vitro and in vivo. As compared to polyclonal Tregs, all 3 manufacture strategies increased the precursor frequency of alloreactive Tregs, and this was proportional to the overall in vitro immunosuppressive properties of the cell products. Accordingly, CAR-Tregs, which contained the highest frequency of donor-reactive Tregs, exhibited the strongest suppressive effects on a cell-per-cell basis. Similarly, in an in vivo mouse model of GvHD, infusion of CAR-Tregs conferred a significantly longer recipient survival than any other Treg product. Our results highlighting the alloantigen-reactivity and associated immunosuppressive properties of different manufactured Treg products have implications for the mechanistic interpretation of currently ongoing clinical trials in transplantation.

Dysregulated anti-oxidant signalling and compromised mitochondrial integrity negatively influence regulatory T cell function and viability in liver disease.

BACKGROUND: Dysregulated inflammatory responses and oxidative stress are key pathogenic drivers of chronic inflammatory diseases such as liver cirrhosis (LC). Regulatory T cells (Tregs) are essential to prevent excessive immune activation and maintain tissue homeostasis. While inflammatory cues are well known to modulate the function and stability of Tregs, the extent to which Tregs are influenced by oxidative stress has not been fully explored. METHODS: The phenotypic and functional properties of CD4+CD25+CD127lo/- Tregs isolated from patients with LC were compared to healthy controls (HC). Treg redox state was investigated by characterizing intracellular reactive oxygen species (ROS), NADPH oxidase-2 (Nox2) activity, mitochondrial function, morphology, and nuclear factor-erythroid 2-related factor (Nrf2) antioxidant signalling. The relevance of Nrf2 and its downstream target, Heme-oxygenase-1 (HO-1), in Treg function, stability, and survival, was further assessed using mouse models and CRISPR/Cas9-mediated HO-1 knock-out. FINDINGS: Circulating Tregs from LC patients displayed a reduced suppressive function, correlating with liver disease severity, associated with phenotypic abnormalities and increased apoptosis. Mechanistically, this was linked to a dysregulated Nrf2 signalling with resultant lower levels of HO-1, enhanced Nox2 activation, and impaired mitochondrial respiration and integrity. The functional deficit in LC Tregs could be partially recapitulated by culturing control Tregs in patient sera. INTERPRETATION: Our findings reveal that Tregs rely on functional redox homeostasis for their function, stability, and survival. Targeting Treg specific anti-oxidant pathways may have therapeutic potential to reverse the Treg impairment in conditions of oxidative damage such as advanced liver disease. FUNDING: This study was funded by the Wellcome Trust (211113/A/18/Z).