Combination antagonism of TNF superfamily signaling for T cell immunosuppression.

INTRODUCTION: The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies comprise 47 proteins that regulate immune signaling and T cell costimulation. While TNF inhibitors are established therapies for immune-mediated inflammatory diseases (IMIDs), their efficacy is limited by primary non-response and secondary loss of efficacy. Preclinical data suggest that the TNF/TNFR members exhibit redundant and synergistic signaling, motivating combination targeting strategies. METHODS: We have systematically evaluated TNF/TNFR combinations as potential immunotolerance targets using integrated computational and experimental approaches. We applied a gene prioritization framework incorporating transcriptomics, genetics, druggability, and pathway regulation data to derive disease association scores for the TNF/TNFR genes in rheumatoid arthritis and inflammatory bowel disease. Based on these scores and T cell expression profiling, ten targets were prioritized for a combinatorial screen using clinical-stage and preclinical pharmacological inhibitors in a mixed lymphocyte reaction (MLR) assay. The effects of the most promising combinations were further characterized by RNA sequencing. RESULTS: The combinatorial screen identified four drug combinations that significantly suppressed T cell production of IL-2 and IFN-γ: TNF+CD40L, TNF+OX40L, CD40L+OX40L, and CD40L+LTβ/LIGHT. Transcriptional profiling revealed that these combinations broadly downregulated genes associated with T cell activation, proliferation, differentiation, and cytokine production that were induced during the allogeneic response. Notably, the co-inhibition of TNF and CD40L (Adalimumab+Dapirolizumab) produced the most robust suppression, uniquely downregulating 337 genes enriched for key T cell activation pathways, including NF-κB and ERK1/2. DISCUSSION: These findings demonstrate that the combinatorial antagonism of TNF/TNFR superfamily members can potently suppress allogeneic T cell responses, with the TNF+CD40L combination showing particularly strong and broad effects. The results support the continued preclinical evaluation of combinatorial TNF/TNFR inhibition as a potential tolerance-inducing therapeutic strategy for patients with refractory IMIDs.