Distinct roles of CD4⁺ and CD8⁺ Tregs in regulating human autoreactive T cells, B cells, and antibodies in genetically engineered tonsil organoid system

Abstract CD4 +and CD8 +regulatory T cells (Treg) are critical for maintaining immune tolerance, and alternations in number and function are associated with detrimental outcomes in infectious and autoimmune diseases. Despite their importance, their distinct roles and mechanisms in regulating self-tolerance need to be better characterized in humans. Here we analyzed the relative contribution of CD4 +and CD8 +Tregs to control autoreactive T cells, B cells, and antibodies in an entirely human tonsil organoid system in vitro. We disrupted their suppressive functions by knocking out FOXP3 and GZMB genes using CRISPR/Cas9 technology. Normally tonsil organoids make robust antibody and cellular responses to live attenuated flu vaccine, but not to autoantigens. In contrast, FOXP3 KO tonsil organoids produced autoantibodies when stimulated with a panel of classical autoantigens, including double-stranded DNA and others. We also detected increased CD8 +T cells specific for autoantigens, including SMCY antigen. With the GZMB KO, we found a marked increase in follicular helper T cells and autoreactive CD4 +and CD8 +T cells. Interestingly the GZMB KO tonsils showed an expansion of plasmablasts, but only a low level of autoantibodies and autoreactive B cells was detected. This indicates that CD8 +Tregs control the early T and B cell activation but not autoantibody production. Moreover, knocking out FOXP3 generated high-affinity HA-specific antibodies in tonsil organoids compared to control, indicating that CD4 +Tregs are a key checkpoint for high-affinity antibodies and the production of autoantibodies. We conclude that CD8 +and CD4 +Tregs have distinct and complementary roles in regulating cellular and humoral responses and preventing autoimmunity.