<p>Here we analyzed the relative contributions of CD4⁺ regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8⁺ regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. <i>FOXP3</i> and <i>GZMB</i> respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4⁺ and CD8⁺ regulatory T cells. Using CRISPR–Cas9 gene editing, we were able to achieve a reduction of ~90–95% in the expression of these genes. <i>FOXP3</i> knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, <i>GZMB</i> knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8⁺ regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8⁺ and CD4⁺ T cells. These findings highlight the distinct yet complementary roles of CD8⁺ and CD4⁺ regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.</p>