CRISPR/Cas9-mediated RELA and RELC knockout in human regulatory T cells abrogates FOXP3 expression and suppressive function

• RELA/B/C knockouts did not alter cytokine production by CD4 + /CD8 + T cells. • RELA / C knockout reduced FOXP3 expression and immune-suppressive function of Tregs. • RELB knockout did not result in loss of human Treg function. • RELC knockdown reduced PD-L1 expression in T cells/Tregs. Mutations in NF-κB-related molecules result in combined immunodeficiency characterized by recurrent infection. In this study, we aimed to investigate the association between mutations in NF-κB family members, RELA, RELB, and RELC, and regulatory T cell (Treg) function in humans. RELA, RELB , and RELC were knocked out using CRISPR/Cas9-mediated homologous recombination in CD4 + /CD8 + T cells and Tregs isolated from healthy donors. The RELA, RELB , and RELC knockouts did not alter the phenotype or cytokine production profile of CD4 + /CD8 + T cells or Jurkat cells. Similar to that observed in knockout mice, RELA or RELC knockout in MT-2 cells and freshly isolated Tregs reduced FOXP3 expression and the immune suppressive function of Tregs. Additionally, PD-L1 expression in effector T cells and Tregs decreased considerably following RELC knockdown. These findings demonstrated that the deletion of RELA or RELC resulted in the loss of Treg-like phenotype and function owing to the downregulation of FOXP3 expression.