Unravelling the immunomodulatory roles of therapeutic tolerogenic dendritic cells in rheumatoid arthritis

Abstract

Tolerogenic dendritic cells (tolDC) hold great promise for the treatment of autoimmune diseases and have shown safety in clinical trials, including the Autologous TolDC for Rheumatoid Arthritis (AuToDeCRA) study. Current research focuses on their efficacy and optimal route of administration. However, there is a critical need for effective tools to monitor therapeutic effects and identify biomarkers associated with successful immunomodulation by tolDC. A hallmark in most individuals with rheumatoid arthritis is the loss of immune tolerance to self, with consequent activation and expansion of pathogenic auto-reactive T cells. Modulating this autoreactive population with the benefit of restoring lost tolerance is an attractive option to specifically target the disease. In my research, I investigated the modulatory effects of tolDC and explored biomarkers reflective of re-established tolerogenicity, to monitor responses to tolDC treatment. I first examined the safety and indirect immunogenicity of in vitro generated tolDC using a skin explant toxicity model, called Skimune®, as part of a secondment at Alcyomics Ltd. This autologous skin model, initially developed to test graft-versus-host reactivity, is now applied to assess the toxicity and immune reactivity of chemical compounds, including advanced therapy medicinal products. My findings showed that T cells primed by autoantigen-loaded tolDC and incubated with skin sections from the same donor did not induce damage according to the Lerner histopathology grading, and inflammatory cytokines such as interferon-gamma were significantly decreased compared to T cells primed with immunogenic mature DC (matDC). Importantly, tolDC were efficient at reducing the damage caused by matDC. Next, I assessed the in situ immune landscape of synovial biopsies from the AuToDeCRA trial, collected before and after tolDC treatment. Using imaging mass cytometry, I validated a panel of 29 markers to stain and analyse paired biopsies. Results revealed a significant increase in myeloid populations expressing high levels of MERTK, a molecule with roles in suppression of inflammation and clearance of apoptotic cells, in all biopsies after treatment, including those from control individuals who received only a saline washout of the joint but no tolDC. The washout procedure could have impacted the balance in cell identities in the joint Autoreactive T cells in rheumatoid arthritis are found in synovial joints effusions, the main site of inflammation, as well as circulating in the blood. Their low frequency poses a challenge for isolating and characterising this population ex vivo, with efforts focused on analysing in vitro clonally expanded subsets, following antigen challenge. In the final part of my project, I explored the feasibility of expanding and sorting flu-specific CD4+ T cells following priming with the flu matrix protein 97 peptide-loaded tolDC or matDC. MP97 is a recall antigen and can be used as a model peptide for proof-of- concept studies. Using MHC class II dextramers, I sorted CD4+ T cells for single-cell RNA and T cell receptor (TCR) sequencing. Transcriptomic data indicated that both tolDC and matDC expanded a Th1 subset, however the proportional abundance was significantly decreased with tolDC versus matDC. Furthermore, this subset showed differential expression of immunosuppressive genes (e.g., increase in Galectin-1 and LAG-3) and pro-inflammatory genes (e.g., downregulation of Lymphoid enhancer binding factor 1 and Linker for activation of T cells) when comparing tolDC- and matDC-expanded Th1 cells. TCR analysis showed that the Th1 subset, as well as a highly proliferating Th1 subtype (Th1-active) were clonally enriched in all conditions, with the Th1-active subset prevalent among CD4+ T cells primed with either tolDC or matDC. Altogether, these findings demonstrate that tolDC prime selected CD4+ T cell subsets towards an anti-inflammatory state, expand a significantly lower amount of Th1 cells compared to matDC, and this subset displays a distinct phenotype and gene expression pattern. Studying antigen-specific T cells remains challenging, but future efforts focusing on the overall impact of tolDC therapies can potentially provide deeper insights into their modulatory effects.