Background: Dendritic cells (DCs) are well-recognized for their dual role either for T cell activation (1) or for inducing T cells tolerance (2). Their ability to modulate T-cell responses has made them an interesting tool for the immunotherapy of autoimmune diseases (3). Cytotoxic T lymphocyte antigen 4 (CTLA4) is a negative co-stimulatory molecule, which binds to CD80/CD86 on DCs. CTLA4 induces its immunoregulatory function through trans-endocytosis resulting in impaired co-stimulation (4), or through the induction of indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme (5). Moreover, it has been demonstrated that CTLA4 impairs the autophagic machinery of DCs and therefore suppresses DC inflammatory function (6). Nevertheless, the molecular mechanisms underlying the CTLA4-mediated immunomodulatory phenotype, require a more comprehensive understanding.

Objectives: In this study we focused on tolerogenic DCs (tolDCs) and we applied CTLA4-Ig as a tool to induce them. We aim to assess the immunoregulatory potential of CTLA4-mediated tolDCs and to investigate thoroughly the intracellular pathways that are involved in the induction of tolerance.

Methods: Healthy human monocytes were isolated from peripheral blood and differentiated into monocyte-derived dendritic cells (DCs). After 6 days, immature DCs activated with LPS were treated with CTLA4-Ig or IgG control for 18 hours. The anti-inflammatory function of DCs was validated using RT-PCR and flow cytometry and DCs proceeded to RNA sequencing. The metabolic pathways were studied using a Seahorse bioanalyzer.

Results: CTLA4-Ig-treated DCs showed significantly decreased HLA-DR, CD80/CD86 expression as compared to IgG-treated cells (n=4, p=0,0294, n=5 p=0,0079). Moreover, IL6 and TNFα mRNA expression, hallmarks of inflammatory cytokines secreted by DCs, was reduced upon CTLA4-Ig (n=5, p=0,0079). To elucidate the pathways involved in DC reprogramming upon CTLA4-Ig treatment, we performed RNA sequencing and we concluded with 1270 differentially expressed genes (p-value <0.05 counts>10). Interestingly, transcriptomic analysis revealed that the majority of genes (n=900) participated in metabolic processes, specifically in OXPHOS pathway and mitochondrial function. To further support the above metabolic changes, we performed Seahorse assays and confirmed that tolDCs had lower basal OXPHOS and decreased ATP production compared with mature DCs. Furthermore, expression of phosphorylated mammalian target of rapamycin (mTOR) and AKT1, central regulators of metabolism, was increased in CTLA4-mediated tolDCs (n=3, p= 0,0308 and p=0,0347).

Conclusion: Herein we confirmed that CTLA4 restricts the pro-inflammatory properties of activated DCs. RNA-seq analysis revealed that this anti-inflammatory deviation of DCs is characterized by the modification of the expression of genes implicated in cellular metabolism. Metabolic experiments confirmed that CTLA4-mediated tolDCs have reduced OXPHOS and ATP production, whereas, mTOR signaling is upregulated. In future experiments, we will investigate the mechanism that CTLA4 may promote metabolic changes thus contributes to the immunoregulatory phenotype of DCs and could represent a therapeutic target.

REFERENCES:

[1]Van Brussel et al. , Mediators Inflamm 2012 , 690-643 (2012).

[2]B. Pulendran et al. , Nature immunology 11 , 647-655 (2010).

[3]B. E. Phillips et al. , Front Immunol 8 , 1279 (2017).

[4]O. S. Qureshi et al. , Science 332 , 600-603 (2011).

[5]D. H. Munn et al. , J Immunol 172 , 4100-4110 (2004).

[6]T. Alissafi et al. , J Clin Invest 127 , 2789-2804 (2017).

Acknowledgements: This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning» in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432), implemented by the State Scholarships Foundation (ΙΚΥ).

Disclosure of Interests: None declared.