Calcineurin blockade modifies dendritic cell differentiation and induces a tolerogenic phenotype

The Nuclear Factor of Activated T Cells (NFATs) is a family of transcription factors activated by the phosphatase Calcineurin (CN). This pathway holds significant clinical relevance, as drugs inhibiting the CN-NFAT axis exert a potent immunosuppressive effect and are utilized to manage organ transplant rejection reactions. Our research group revealed that NFATs are activated in dendritic cells (DCs), leading to terminal differentiation and apoptosis. Given the immunosuppressive role played by certain types of myeloid cells in pathological contexts, we aim to determine the involvement of the CN-NFAT pathway in early DC differentiation and its potential contribution to the development of an immunosuppressive phenotype in myeloid cells. To explore this, we transduced a growth factor-dependent splenic dendritic cell (DC) line with a CN inhibitor peptide, generating DC-iCN. To determine the effects of NFATs inhibition in DC-iCN, we conducted proteomic analyses, as well as functional and metabolic assays. The potential immunosuppressive activity of the DC-iCN was determined by Mixed Leukocyte Reaction assay. We analyzed the potential effects of inhibiting NFAT in vivo through the intravenous administration of nanoparticles carrying the iCN peptide to investigate whether this would modify the hematopoietic process leading to the formation of DCs. Blocking the CN-NFAT pathway in DCs increased their growth rate and modified the cell cycle, prolonging the G2/M phase and shortening the G1 phase. Metabolically, NFAT inhibition induced a marked Warburg effect, characteristic of rapidly proliferating cells. Moreover, DC-iCN demonstrated a highly immunosuppressive phenotype, differentiating naïve T cells into FOXP3+ regulatory T cells in MLR assays. Interestingly, inhibiting the CN-NFAT pathway in vivo expanded the granulocyte-monocyte progenitor involved in DC ontogeny. In summary, blocking the CN-NFAT pathway modifies early DC differentiation, resulting in the acquisition of a highly tolerogenic phenotype and traits resembling undifferentiated cells. This sheds light on the potential use of tolerogenic hematopoietic precursors as a cellular therapy for autoimmune diseases and transplant rejection. Further human studies will enhance these findings.