DEF6 is a guanine nucleotide exchange factor acting downstream of T-cell receptor stimulation. While murine studies suggest a role in immune homeostasis and possibly in the onset of autoimmunity, DEF6 deficiency has not yet been attributed with human disease.
In three patients from two unrelated kindreds with systemic autoimmune manifestations including lymphocytic organ infiltrates, splenomegaly, bowel inflammation, autoimmune hemolytic anemia, and intermittent lymphopenic episodes, we applied homozygosity mapping and whole-exome sequencing to identify biallelic missense variants in DEF6. We investigated immune cell functions and employed cell biological and biochemical assays to characterize functional DEF6 deficiency in T cells.
We identified impaired regulation of the immune checkpoint protein CTLA-4 as mechanistic consequence underlying human DEF6 deficiency, causing autoimmune presentation. Patient T cells exhibited impaired regulation of CTLA-4 vesicular trafficking associated with reduced CTLA-4 surface availability. Cycling defects were replicated in DEF6-knockout cell lines. We identified the small GTPase and endosomal recycling marker RAB11 as a novel interactor and in vitro substrate of DEF6, and found disrupted binding of mutant DEF6 to RAB11. RAB11+CTLA-4+ recycling vesicles were absent in DEF6-mutated cells. CTLA-4-Ig treatment was initiated for one of the patients, achieving sustained remission.
We uncovered DEF6 as player in human immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surfaces, with potential significance for autoimmune and/or cancer therapy.