The immune system has various checkpoints to prevent the activation of self-reactive lymphocytes, but the mechanisms by which some lymphocytes bypass these constraints and cause autoimmune diseases are not well understood. A prevailing theory suggests that somatic mutations in immune-regulatory genes might allow self-reactive lymphocytes to evade these checkpoints. However, testing this hypothesis has been difficult due to technical challenges. In this study, researchers employed whole-exome and targeted NanoSeq, a precise single-molecule DNA sequencing method, to investigate driver mutations in autoimmune thyroid disease. The study uncovered numerous B cell clones with loss-of-function mutations in critical immune checkpoint genes, such as TNFRSF14 (HVEM) and CD274 (PD-L1), along with less frequent mutations in other immune genes. In highly inflamed tissue samples, tens to hundreds of independent immune checkpoint mutant clones were identified. Techniques like laser microdissection, methylation sequencing, spatial transcriptomics, immunostaining, single-nucleus DNA sequencing, and antibody synthesis were used to localize these mutations to B cells, confirm their self-reactivity, and identify clones with multiple mutations. The research revealed widespread biallelic loss of TNFRSF14 and clones with up to 4-6 driver mutations, although each clone represented only a small fraction of the total cell population.
